Sets of Giemsa-stained, blood smear slides with systematically verified composite diagnoses would contribute substantially to development of externally validated quality assurance systems for the microscopic diagnosis of malaria.Plasmodium-positive donors in Cambodia and Indonesia and individuals with no history of risk for malaria was collected. Using standard operating procedures, technicians prepared Giemsa-stained thick and thin smears from each donor. One slide from each of the first 35 donations was distributed to each of 28 individuals acknowledged by reputation as having expertise in the microscopic diagnosis of malaria. These reference readers recorded presence or absence of Plasmodium species and parasite density. A composite diagnosis for each donation was determined based on microscopic findings and species-specific small subunit ribosomal RNA (ssrRNA) DNA polymerase chain reaction (PCR) amplification.whole blood from Plasmodium falciparum (99%), followed by Plasmodium vivax (86%).More than 12, 000 slides were generated from 124 donations. Reference readers correctly identified presence of parasites on 85% of slides with densities <100 parasites/μl, which improved to 100% for densities >350 parasites/μl. Percentages of agreement with composite diagnoses were highest for Herein, a standardized method for producing large numbers of consistently high quality, durable Giemsa-stained blood smears and validating composite diagnoses for the purpose of creating a malaria slide repository in support of initiatives to improve training and competency assessment amidst a background of variability in diagnosis is described. Plasmodium falciparum and other species, accurate species detection becomes critical [Despite advances in antigen detection and gene sequence amplification technology, microscopic examination of Giemsa-stained blood film is still the gold standard for malaria diagnosis. Early malariologists, including Ronald Ross, emphasized the importance of externally validating results and training microscopists . Decadescritical  and the The quality of microscopy is highly variable and methods for validating proficiency are not standardized. Currently described methods for slide preparation ,6,7 yielMost recommendations for quality assurance procedures in microscopy date back to the days of global eradication efforts. In countries where malaria eradication programme failed, skills at the healthcare periphery, where the vast majority of patients seek medical care, and within the reference laboratories themselves, have not been adequately sustained. The emergence of the new malaria control paradigm, focusing on treatment rather than prevention , demandsThe lack of acknowledged standards of competency and a method for ascertaining individual competency imposes one of the primary obstacles to reliable diagnosis. Production of a collection of Giemsa-stained blood films, each having meticulously vetted composite diagnoses, as described herein, is the first step in addressing this problem by. This collection offers a true "gold standard" for assessments of reader competency as well as sensitivity and specificity for other diagnostic modalities. Such rigor can serve as the basis for externally validated competency at laboratories responsible for compiling and distributing blood films for international, national, local and certification programmes.Donors were selected to support the primary objective of creating a slide repository. In Cambodia, adults attending health centers with acute fever served as the sources of donors. In Indonesia, people participating in Institutional Review Board (IRB)-approved research protocols or in malariometric surveys volunteered to participate. All donors positive for plasmodia were treated immediately following donation of blood according to prescribed practice in each setting or according to the IRB-approved research protocols in which they were participating. Unambiguously malaria negative donors were restricted to natives of a malaria-free country without travel to a malaria endemic area for the past two years or donating blood within five days of any possible exposure to risk of infection, e.g., a person arriving in Indonesia from the United States. Project protocols received ethical review approvals by the National Ethics Committee for Health Research (NEC) of the Cambodian Ministry of Health and the National Institute of Health Research and Development of the Indonesian Ministry of Health. After donors provided informed consent, technicians collected 3 cc of whole blood, transferred it to an EDTA-containing glass test tube and mixed it by gentle inversion.® Rite-on superthin microslides, 75 × 25 mm). Technicians washed all new slides by soaking them in a tub of soapy water for at least 2 hours. After rinsing with tap water, each slide was individually dried with cotton gauze or a lint-free towel and placed on a hot plate (50–60°C) until completely dry. Slides were stored in plastic boxes containing silica gel. Just prior to blood collection, the donor's sequential identity number was written in pencil on the frosted end of each slide. Blood was always placed on the slides within 6 hours of collection. Technicians never processed slides from more than one donor at any given time and never more than three donors per day.Smears were prepared on pre-cleaned and uncoated glass slides with frosted ends  Merck Giemsa Solution  was prepared just prior to staining each set of slides. After overnight drying, technicians fixed thin smears by dipping in a container of absolute methanol, avoiding contact between methanol and the thick film. After a few minutes drying, slides were arranged in a staining tray for batch Giemsa staining (maximum 50 slides per batch). After 30 minutes at room temperature, technicians removed the slide trays from the Giemsa solution and gently swirled them in a container of tap water before placement in a slotted plastic or wooden block for drying at room temperature.®  mounting medium, 1–2 ml transferred to a clean glass test tube (single use for each session). Using an Eppendorf pipettor, technicians transferred 100 μL of Poly-Mount® to each slide as a thin line running lengthwise from the frosted end to the feathered edge of the thin film, without allowing the pipette tip to touch the stained blood film. To minimize air bubbles under the cover slips , technicians gently lowered them to the surface of the slides from roughly a 45° angle slowly into the horizontal position. Technicians then applied gentle pressure to the cover slips to spread mounting medium evenly and wiped excess mounting medium from the edges of the slides. Slides dried in ambient air overnight before labeling and storage in slide boxes. Each slide was labeled with a unique encrypted 8 digit identifier, which was printed in numeric and barcode format  so that it could not be linked to a specific donor by readers.Technicians placed cover slips over the thick and thin smear to increase shelf life. Once the Giemsa-stained slides dried, they were stored in clean, plastic slide boxes. Within two weeks, technicians prepared them for batch cover-slipping using Poly-Mount® 100 5% resin  in distilled water. Samples were incubated for 20 minutes on a 56°C heat block followed by eight minutes on a 100°C heat block. Diagnostic PCR amplification of the gene for small-subunit ribosomal RNA (ssrRNA DNA) was conducted using the methods of Kimura et al. [At the time of venous blood sampling, blood blots were collected onto Whatman No. 1 filter paper  for drying and storage. Any whole blood remaining after slide preparation was stored at -70°C. DNA was extracted from dried blood blot samples using Chelexa et al.  to detecAfter obtaining a provisional microscopic diagnosis by staff microscopists, 30 slides from each of 35 donors, without providing the diagnoses, were referred for reference reader evaluation and ultimately, determination of a composite diagnosis for each donor blood sample. After soliciting referrals from experts in the malaria research community, 43 individuals were invited to participate in the study as reference readers. All twenty-eight (65%) who agreed to participate were selected as reference readers. They represented 13 countries on five continents. Confidentiality of the reference readers was guaranteed and all understood they were among a pool of other reference readers unknown to them. Reference readers were aware that positive slides were collected in Cambodia and Indonesia but were unaware of what proportion of the slides they reviewed was positive. Slides sent to each reference reader contained one slide from each of the 35 donations.Reference readers were provided guidelines for slide reading methodology and asked them to record presence of malaria parasites, species, stages, and parasite counts using a standard electronic form that automatically calculated the parasite density (per μl) based on the total number of asexual stage parasites and WBC counted in the high power thick film fields and a standard multiplier of 8,000 WBC/μl. Although the method for enumerating parasites was not mandated, the WBC method  was recoReported results of the reference readers were compiled to complete two distinct tasks: 1) identifying unqualified reference readers with consistently incompatible results relative to other readers and 2) deriving a composite diagnosis for as many of the donations as possible based on analysis of the combined results of the reference readers and PCR analysis of the particular donation. The composite diagnosis was to be accepted as the true diagnosis. The objective was simply to assemble a collection of blood films for which composite diagnoses could be accepted as accurate. In the event of a high degree of discordance among readers for a particular donation, those slides were excluded as inherently ambiguous and not suitable for use in the slide sets. The process of both qualifying reference readers and establishing a composite diagnosis among them is described below.P. falciparum parasitaemia infection by P. vivax) or partially correct by degree when dealing with mixed infections with one species having an extremely low count. Finally (tertiary), the parasite counts were considered, for which individual accuracy was ascertained by accepting the median count among qualified readers as the best estimate of true count. A primary diagnosis was accepted as the composite diagnosis with >80% accordance among readers. Among positive smears, the composite secondary diagnosis required >80% accordance on single species diagnoses. When readers identified a mixed infection, even if numerically in the minority, and that secondary diagnosis was affirmed by PCR analysis, their view was accepted as the composite diagnosis. When PCR analysis detected the presence of a species not identified by any of the reference readers, the composite diagnosis was assigned based on microscopic observation alone, and the PCR result was attributed to a second infection below the limit of microscopic detection.Three tiers of diagnostic proficiency were established and classified by assigning demerit points based on types of errors made Table . First  slides were discarded on the basis of poor quality. Based on availability and slide set requirements, the first 35 of the 124 donations were selected for the rigorous process of external validation by composite diagnosis among reference readers.P. falciparum, P. vivax and P. malariae mono-infections (secondary level of diagnosis) 99%, 86% and 50% of the time, respectively. Mixed infections were identified in only 47% of the time, usually a consequence of low density parasitaemia of the second species not reported. This observation was most notable for donation 12 . The findings of 4 readers with demerit points greater than one standard deviation above the mean from further analysis were excluded , five P. vivax , three mixed P. falciparum/P. vivax  and two P. malariae . Composite diagnoses were not determined for the seven remaining donations because the reference reader results were equivocal . Three of the donations,  were not used for the slide sets because the densities and species they contained were not required. Donation 12 represents the sole deviation from composite diagnosis selection criteria outlined in Table P. vivax co-infection with P. falciparum, a composite diagnosis of mixed P. falciparum/P. vivax infection was assigned because: 1) the accuracies of the three reference readers who identified the P. vivax were exemplary for all other slides ; 2) it is likely that due diligence in identifying the second species on the part of the other 21 microscopists was obviated by presence of P. falciparum at such a high density  and 3) the presence of P. vivax DNA was confirmed by PCR.Based on the 24 reference reader results, a composite diagnosis was derived, including parasite density, for 28 of the 35 evaluated donations , 18% lower density P. falciparum , 10% higher density P. vivax , 9% lower density P. vivax (≤300/μl), and 19% mixed P. falciparum/P. vivax and P. malariae slides. These slide sets are now available for distribution to MR4 registered users for a limited use period. Copies of the standard operating procedures (SOPs) used for their creation are also available from MR4.From the slides with composite diagnoses, 24 sets of 50 slides each were compiled and provided to the U.S. National Institutes of Health Malaria Research and Reference Reagent Resource Center  with answer keys containing the median parasite densities and 99% CI as a guide for acceptable quantification. Sets contained slides with the following distribution: 25% negative, 19% higher density Using the currently available standard of competency for microscopic diagnosis of malaria, namely reputation, a team of reference readers was assembled and by critically evaluating their performance relative to one another and PCR-based evidence, their level of competency was affirmed. The product of this exercise, the collection of standardized Giemsa-stained blood films described, is as nearly unambiguous as possible with respect to accuracy of diagnoses. This collection, or others similarly prepared, would provide a validated, objective benchmark for evaluating the competency of microscopists in research, clinical and public health settings or the validity of other diagnostic methods.P. malariae) and mixed infections are more difficult. Ultimately, if diagnostic proficiency is to be objectively compared to raise international standards of performance, a slide bank such as the one described here would serve as the only viable means of such a fair comparison, systematically using the same blood with the exact same degree of difficulty.It is intuitively obvious that the difficulty of any test is dependent upon the ease of the questions being asked, an observation that holds true for practical assessment of proficiency in the microscopic diagnosis of malaria. Even among the panel of experts assembled for this project, rates of agreement with the composite diagnosis were closely tied with the difficulty of the slide under evaluation, presuming that slides with lower density parasitaemia, less commonly encountered species  and mixed infections are more difficult. A mean 15% failed to detect parasites when densities were less than 100/μl and only 47% (range 13–67%) accurately diagnosed mixed infections.The findings described here highlight and confirm many of the previously published difficulties facing diagnosis of malaria today. Milne et al.  found thPlasmodium-positive and negative blood smears was precluded [The standard operating procedures for this project were derived in an attempt to resolve some of the inherent imprecision associated with the variability in methodology for slide preparation and parasite quantification including the use of precise blood volumes and a thick film template to ensure consistency in blood film thickness -18. Althrecluded ,21. PermSome methods for quantifying parasitaemia may be cumbersome . Using tNo standards currently exist for determining what constitutes competent microscopy, let alone an expert microscopist. The experience described herein with 28 reputed experts, specifically the variability in results observed, highlights this point. Such standards must be established. Production of durable, validated high quality standardized malaria microscopy slides is the first and essential step toward that end. Using the methodologies described herein, institutions from around the world could establish repositories of uniform slide sets to meet the growing demand for verifiable training, testing and quality assurance materials. Ultimately, if diagnostic proficiency is to be objectively compared to raise international standards of performance, a slide bank such as the one described here serves as the only viable means of such a fair comparison, systematically using the same blood with the exact same degree of difficulty.JDM coordinated project planning and implementation in Indonesia and Cambodia and protocol development at both locations, supervised execution and data management and wrote the manuscript. ERL was the principal investigator in Indonesia, responsible for project execution. MJB coordinated efforts of the expert readers, and was directly involved in data entry and management. WAP conducted statistical analysis. RGJ contributed to project planning and compilation of the microscopist panel. SD was involved in execution of the project in Cambodia.SM was involved in execution of the project in Cambodia. PS was involved in execution of the project in Indonesia. MJB participated in project planning oversight in Indonesia. WRP participated in project planning and oversight of microscopist team and data flow. JKB provided expert consultation in all phases of project. CW was the principal investigator in Cambodia, responsible for project execution. All authors read and approved the final manuscript.The assertions herein are the views of the authors and do not reflect official policy of the U.S. Department of the Navy or the U.S. Department of Defense.


Neisseria meningitidis (serogroup C in 1979 and B in 1987), and Haemophilus influenzae type b (1999), two of the main causal pathogens. We described and discussed some epidemiological aspects of the current status of bacterial meningitis to learn from the Cuban experience.Bacterial meningitis is a group of life threatening infections that mostly affect children and adolescents, and may be the cause of severe neurological sequelae. Cuba has implemented massive vaccination programmes against both A nationwide observational study on children and adolescents from 1 to 18 years old was carried out from 1998 to 2003, estimating the incidence and case-fatality rate by age group and causal pathogens, as well as the seasonality and frequency of overcrowded dormitories. The association between disease and attendance to day care centres or boarding schools was estimated by using relative risk (Chi-squared test and Fisher Exact Test).Streptococcus pneumoniae, Haemophilus influenzae type b and Neisseria meningitidis serogroup B were the main identified agents. The average case-fatality rate was 10.5% and the most lethal agents were Streptococcus pneumoniae (27%) and Haemophilus influenzae type b (10.7%). Overall percentage of cases who slept in overcrowded dormitories was 15%, reaching 30.6% in adolescents. Seasonality was only evident among meningococcal meningitis cases between September–October. The attendance to boarding high school showed an association with disease only in 1998 and 1999 .The overall number of cases was 1023; the incidence ranged from 3.4 to 8.5 per 100 000 population, with the higher figures in children 1–5 years old (16.8 per 100 000 population). Neisseria meningitidis serogroup B and C and Haemophilus influenzae type b through a national immunisation program.The highest incidence of bacterial meningitis was observed among children from 1–5 years old. Pneumococcus was both the leading causal and the most lethal agent. Sleeping in overcrowded dormitories was more frequent among adolescents. No strong association was observed between the bacterial meningitis and attendance to day care centres or boarding schools. The incidence of bacterial meningitis in Cuba is declining after massive vaccination programmes against  Bacterial meningitis (BM) is a group of severe infections that frequently affect children and adolescents, carrying a high case-fatality rate (CFR). It may cause transient or permanent deafness as well as other severe neurological sequelae in survivors -3.Neisseria meningitidis, Haemophilus influenzae type b (Hib) and Streptococcus pneumoniae (Spn) are the main triad responsible for more than 80% of all cases. Gram negative bacteria, particularly E. coli, Streptococci (different from S. pneumoniae), Listeria monocitogenes and Staphylococci may also cause BM [The World Health Organisation estimated that annually BM causes at least 1.2 million cases worldwide and of those 135 000 deaths. Though other bacteria may cause BM, th century [BM has been reported in Cuba since the beginning of the 20 century . The manFollowing a meningococcal disease epidemic in 1979, an independent surveillance system was put into place . After cNeisseria meningitidis serogroup C (1979) and B (1987) [On the other hand, Cuba is the only country in the world which has carried out massive vaccination initiatives against group C 179 and B B (1987) , both asThe aim of this study was to describe and discuss the results of both, the BMSS and the massive vaccination strategies, on the epidemiology of BM in Cuban children and adolescents.st 1998 to December 31st 2003 were included in the study, considering the date of the symptoms onset.1023 cases of BM in the Cuban population between 1–18 years old reported nationwide by BMSS from January 1Children under 1 years old were not included in the study based on the criteria that they should be considered a special group for the following reasons: breast-feeding, immune maturation, they usually do not attend day care and do not have much community contact.We defined a case of BM as "a clinical meningeal syndrome, through the identification of the causal agent directly by culture blood, petecchias, cerebrospinal fluid (CSF) analysis or indirectly by polymerase chain reaction, latex test or another rapid diagnostic test. The cases with negative culture or bacteriological test, but the cyto-chemical exam of the CSF suggested bacterial infection, were considered BM of "unknown aetiology" .A total of 4 age groups which coincided with the main level of education in Cuba were selected: the group 1–5 year olds which attended day care centres (DCC) was considered as small children; 6–11 year olds were considered older children (Primary School). More than 11 year olds were considered adolescents, attending High School those 12–14 year olds and to College, Technical or Professional Education those 15–18 year olds.The incidence rate (new cases per 100 000 population) and CFR (percent) were calculated according to the age group and to the main causal agent, using the data of estimated Cuban population of the Office for National Statistics in Cuba. The seasonality by monthly average for each causal agent and the frequency of cases who slept in overcrowded dormitories by age group were also estimated. Comparisons were carried out using Chi-squared tests with a significance level of 0.05.Relative Risk (RR) was estimated and its confidence interval with p < 0.05 considered as significant, using either a Chi-squared Test or Fisher's Exact Test as appropriate. We considered exposed to community settings the cases attending DCC or boarding school, and not exposed those who do not attend these institutions. The national matriculation and registration of school children and adolescents during each school year were obtained from the Ministry of Education (MINED). RR in primary school children was not estimated because the boarding student matriculate between 1998–2003 was not available.a) and Excel (version 5.1) for the statistical analysis, and Microsoft Word 2000 as the text processor program.We used Epi-Info , with a subsequent decrease. The group 1–5 year olds showed the highest incidence of all.  over female (46.8%) was observed in all the groups (data not shown).N. meningitidis were the main causal pathogens identified.Spn, Hib and ®). The greatest decrease of incidence was observed in small children, from 10.7 per 100 000 population in 1999 to 5.7 per 100 000 population in 2000 (p < 0.05). Only 8 cases have been reported since 2002 (Table In 1998 Hib showed the highest incidence (3.0 per 100 000 population), but dropped significantly by approximately half (1.6 per 100 000 population) after the vaccination programme in 1999 with a conjugated vaccine  and subsequently decreasing. A similar pattern in the incidence was observed in all age groups with the exception of adolescents from 15–18 years old. The small children showed the highest incidence of all groups, mainly in 1998 (2.8 per 100 000 population) and in 1999 (2.9 per 100 000 population), decreasing in 2001 and 2002, with a discreet subsequent increase in 2003 (2.3 per 100 000 population). Since the year 2000 Spn has shown the highest incidence of all the identified bacteria .Staphylococcus sp., Streptococci (different from S. pneumoniae), E. coli and Salmonella were the most common pathogens. The frequency of this group of bacteria was 43% in small children, 28% in the older children, 13% in adolescents from 12–14 year olds and 16% in adolescents from 15–18 year olds. Gram-positive bacteria (63%) was significantly more frequent than the gram-negative (37%) (Data not shown).Regarding other identified bacteria, The average CFR of BM was nearly 10.5%. Overall CFR showed variations, increasing significantly in 2000 (13.6%) and in 2001 (13.6%) when compared to 1998 (7.8%), with a subsequent significant decrease in 2003 (10.9%) , followed by Hib with CFR = 10.7% (8–75%) and Meningococcus B with CFR = 4% (4.7–10.5%). The highest CFR (75%) observed in the period was caused by Hib in 2001, but no cases have been reported subsequently  to 1999 (4.8 per 100 000 population), as well as by age group. Only among small children a significant decrease was observed, from 4.6 per 100 000 population in 1998 to 2.2 per 100 000 population in 2003  (Data not shown).No association of BM with attendance to DCC or boarding school  was observed during the study. Only in 1998 and 1999 did the attendance to boarding high school show a strong association with disease  Table .Prevention and control of infectious diseases, especially among children and adolescents, has been a big concern for the Public Health Ministry (PHM) in Cuba. Hitherto vaccines are the most promising prospect for the prevention of community acquired BM. Therefore widespread use of effective vaccines may induce a major impact in preventing the disease, especially when they are used during a long period of time as a part of the NIP, as occurred in Cuba, where a decreasing trend of BM has been documented after vaccination against meningococcus serogroup B and C, and more recently, Hib ,9,10,12.Cuban strategy to prevent these infections has been an initial vaccination campaign continued through the NIP.The first intervention was carried out in 1979 with an A+C vaccine (Pasteur-Merieux) during the meningococcal disease epidemic caused mainly by serogroup C  and B 34,3%). The target population was 3, 245 046 individuals from 3 months of age to 19 years old, considered at high risk of disease, achieving 78.2% vaccination coverage. This intervention decreased significantly the serogroup C (7.2%) in 1980, but was ineffective against serogroup B, therefore the incidence continued to rise, but having serogroup B as a predominant causal agent (78.4%) ,3%. The .®) was first administered to high risk age groups and subsequently to all the population from 3 months of age to 24 years of age (an estimated 2 million people or more) [By the end of 1988, after having being evaluated in a double-blind, placebo-controlled trial, the Cuban anti-meningococcal BC vaccine (VA-MENGOC B-Cor more) .Neisseria meningitidis  has been the only one isolated bacteria and subsequently confirmed by the Neisseria Laboratory at the IPK, where the strain B4. P1. 19.15 has been the most frequently identified, using ELISA [During this period, serogroup B ng ELISA . It is lShifts in the frequency and circulation of each bacteria and/or strain into a region, country or continent depends on the interaction of both the pathogen and the host within settled environmental conditions, nevertheless important changes may be induced by prolonged and massive vaccination programmes.Small children are widely considered a high-risk group for BM, as it was confirmed in our study, where the highest incidence was observed in less than 6 year olds. ,16. In CDue to the increase of infant mortality caused by Hib infections, the PHM decided to start using an available conjugate vaccine against Hib in 1999. The vaccine was initially used in a campaign for small children and continued through the NIP . The incFor these reasons, we considered that the vaccination against Hib contributed decisively to the overall decrease of BM in small children described above, and undoubtedly the development of such vaccines has been one of the most important events in the history of the prevention and control of infectious diseases in paediatrics ,20.Spn is not a common cause of meningitis epidemics and outbreaks . We consThe improvement of the BM surveillance system in Cuba increases substantially quantitative and qualitative information about this group of infections . DespiteRegarding the gender, we observed in our study that infections in male predominated those in females, contrarily to other author that point out non significant differences between males and females .CFR depends on, among other factors, the host response, the virulence of the pathogen and the quality and timeliness of medical attention. Average CFR was nearly 10% and similar figures are reported in many developed countries, where it could be as low as 2% and as high as 25% ,24, thouTwo well-defined seasons are recognised in Cuba: the rainy (April to September) and the dry (October to March). The monthly average case distribution of Hib and pneumococcal meningitis, did not show seasonality differences, but meningococcal meningitis did, increasing the number of cases mainly in September, October and March, coinciding with reports of the literature . In CubaAirborne transmission of infections can be favoured by some environmental conditions such as attendance at boarding institutions, overcrowding areas, poor ventilation and intimate contact ,15,27.In our study we observed that the overall percentage of cases sleeping in overcrowded dormitories was 15%, but in adolescents reached nearly 30%. It should be considered as an important risk for the transmission of BM in this group, and must call the attention of both, MINED and PHM, for the avoidance of these conditions, where possible, especially in DCC and boarding school.On the other hand, as a nation-wide improvement of the Cuban educational system during the seventies, a huge number of boarding school  were built nationwide. In these institutions the intimate contact and promiscuity among boarding students may increase airborne infections ,15,27-29Surveillance is a key subject for the control and prevention of infectious diseases and provides information about main epidemiological and microbiological aspects to put in place correct strategies.Massive vaccinations against meningococcus, and Hib have decreased overall BM incidence in Cuba, and showing Pneumococcus as the leading causal pathogen and the most lethal.The disease was more frequently observed among male children under 6 years of age.Seasonality was only observed in meningococcal meningitis. Dry season and the start of the school year may also be contributing environmental conditions.There was a weak association of BM with attendance to DCC and boarding school, and may be the effect of massive and systematic vaccination, which in our opinion are the most adequate and effective measures for the prevention and control of these infections.Further massive vaccination initiatives with available anti-pneumococcal conjugated vaccine  may decrease the incidence of BM even more. Surveillance systems will contribute towards the detection of the emergence of other causal pathogens or any other changes in the epidemiology of these infections.The author(s) declare that they have no competing interests.FDM conceived the study, participated in the design, performed the statistical analysis, and the draft of the manuscript and was the main responsible author.APR participated in the design and draft of the manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:


Surprisingly little is known about the geographical overlap between malaria and other tropical diseases, including helminth infections. This is despite the potential public health importance of co-infection and synergistic opportunities for control.Plasmodium falciparum malaria, adjusted for urbanization, the spatial congruence of populations at coincident risk of infection is determined.Statistical models are presented that predict the large-scale distribution of hookworm in sub-Saharan Africa (SSA), based on the relationship between prevalence of infection among schoolchildren and remotely sensed environmental variables. Using a climate-based spatial model of the transmission potential for The model of hookworm indicates that the infection is widespread throughout Africa and that, of the 179.3 million school-aged children who live on the continent, 50.0 (95% CI: 48.9–51.1) million  are infected with hookworm and 45.1 (95% CI: 43.9–46) million are estimated to be at risk of coincident infection.Malaria and hookworm infection are widespread throughout SSA and over a quarter of school-aged children in sub-Saharan Africa appear to be at risk of coincident infection and thus at enhanced risk of clinical disease. The results suggest that the control of parasitic helminths and of malaria in school children could be viewed as essential co-contributors to promoting the health of schoolchildren. Plasmodium falciparum malaria inflicts the largest burden  is associated with hookworm or malaria infection in Africa is not clear, with studies yielding contrasting results ,43. SuchP. falciparum malaria transmission intensity across Africa are lacking despite long advocacy for their importance [The approach adopted in the present analysis highlights a number of key methodological limitations. First, the errors surrounding our estimates of populations at risk only take into account those arising from fitting of the logistic regression models to the observed hookworm data and, therefore, underestimate the true error of the population-at-risk estimates by an unknown quantity. This is because there are a number of other potential sources of error that are not included in our estimates, ranging from measurement error of parasitological diagnosis and the satellite-derived independent variables, to multiple sources of error in the population density maps. Additionally, as the current models did not incorporate spatial correlation, a known feature of parasitological data, this will have led to further underestimation of true error. Second, there are limitations with the MARA malaria risk map. While the MARA model has enjoyed wide use, it has been validated against parasite prevalence data only in Kenya . Extensiportance . Revisedportance . Third, portance  and urbaportance  in AfricFinally, the extent to which co-distribution across population implies co-infection within individuals cannot accurately be defined on the basis of the current data. Nevertheless, the occurrence of co-infection is probably higher than simple probability would suggest due to the observed clustering of both infections in certain individuals and households ,50. ThisP. falciparum and hookworm. In terms of school health programmes, they also suggest that the control of parasitic helminths and of malaria in school children, interventions which tend currently to be perceived as having separate goals, could in fact be viewed as essential co-contributors to promoting the health of children.The present analysis shows that malaria and hookworm infection are widespread throughout SSA and over a quarter of school-aged children in sub-Saharan Africa appear to be at risk of coincident infection and thus at enhanced risk of clinical disease. The results highlight an important research agenda that includes investigating both the causation and consequences of co-infection with The study was conceived by SB with input from RWS. SB collated the survey data and ACAC developed the statistical models. SIH provided the satellite data and the adjusted malaria risk model, and AJT undertook geographical extractions and analyses of these data. PJH, DAPB and RWS provided epidemiological interpretation of the data and background material. SB wrote the first draft of the paper and all authors contributed to, read and approved the final manuscript.Data used to develop predictive models of hookworm. The prevalence data used to develop the predictive models of hookworm, including data sources and sample sizes.Click here for file


Leptospira serology  was reported positive on day 5. Dual infections are common and under-recognized in the tropics. Failure to treat potential co-infections may lead to poor outcomes. Acute lung injury in falciparum malaria has high mortality rates and therapy as for severe sepsis may improve survival. Adjunctive therapies, including activated protein C, cannot replace source eradication.Co-infection with falciparum malaria and leptospirosis is uncommon. The aim of this study is to report a case of severe sepsis secondary to dual infection with falciparum malaria and leptospirosis. The literature is also reviewed on the clinical course of such co-infections, and the possible mechanisms and treatment of patients with life-threatening malaria and leptospirosis with activated protein C. The patient was a 25-year old male admitted in the Respiratory Intensive Care Unit (RICU) with fever, haemolysis, acute renal failure, hepatitis, acute lung injury (ALI) and altered sensorium. A syndromic evaluation was done and investigations revealed falciparum parasitaemia. He was treated with parenteral artesunate, ceftriaxone and doxycycline, and adjunctive therapies as for severe sepsis. Infusion of activated protein C was started 20 hours after onset of organ dysfunction, and intensive haemodialysis was instituted. Over the next four days the patient became afebrile with progressive resolution of ALI, renal failure and hepatitis. His  The world-wide distribution of malaria  overlaps2/FiO2 score of 205. Chest X-ray revealed bilateral peri-hilar infiltrates suggestive of acute lung injury  had been considered and he had received two doses of intravenous artesunate 120 mg). On examination he was found to be drowsy and febrile; he also had icterus and sub-conjuctival haemorrhages. The respiratory rate was 45 breaths/minute, blood pressure 110/70 mm Hg and heart rate 126 beats/minute. Auscultation of the chest revealed bilateral crepitations and examination of the abdomen showed hepatosplenomegaly. The rest of the examination was unremarkable. Biochemical investigations revealed a serum creatinine of 7 mg/dL, serum bilirubin 35 mg/dL (conjugated fraction 23 mg/dL), serum bicarbonate of 10 mEq/L, plasma haemoglobin (Hb) 24 mg/dL and urine Hb of 10 mg/dL. Complete blood count showed a haemoglobin of 9.6 gm/dL, total leucocyte count of 15,600/μL and platelet count of 98,000/μL). A syndromic diagnosis of infective hepatorenal syndrome was made and he was managed in the respiratory intensive care unit with artesunate (150 mg o.d), ceftriaxone (two gm b.i.d) and doxycycline (100 mg b.i.d), stress ulcer and deep venous thrombosis prophylaxis and intensive blood glucose control. A thin blood smear was positive for falciparum ring forms (1.2%) and gametocytes ; an assay for histidine-rich protein 2 obtained simultaneously was also positive. A clotting profile was normal and his arterial blood gas revealed combined metabolic and respiratory acidosis (pH 7.12) and hypoxaemia with a PaO20 mg. OnThere are about 500 million estimated cases of malaria, with 2–3 million severe malaria cases and 1.1 million deaths each year worldwide . LeptospIn a patient with fever, renal failure and jaundice the differential diagnosis include severe malaria, severe leptospirosis, enteric fever, Hantaan virus, viral hepatitis with fulminant liver failure and scrub typhus ,10. The As malaria and leptospirosis are common in the tropics, co-infections are expected to be common. However such a scenario has seldom been reported ,13. ThisAcute respiratory distress syndrome in falciparum malaria has an incidence of 21% in those with severe malaria requiring hospitalization . MortaliIn patients with severe falciparum malaria, extensive organ sequestration may lead to endothelial clogging and dysfunction. Microthrombosis and vasospasm due to endothelial dysfunction lead to organ hypo-perfusion and worsening lactic acidosis. Adjunctive therapies like exchange transfusion  and antiDual infections are common and under-recognized in the tropics. Clinical features are unreliable to separate single infections from dual infections, which may have a severe clinical course. Both falciparum malaria and leptospirosis are associated with significant endothelial dysfunction and therapies that improve endothelial function might positively impact clinical outcomes of both diseases. aPC has anti-thrombotic, pro-fibrinolytic and anti-inflammatory properties which can improve endothelial function. Despite the potential benefits of this therapy, source eradication with appropriate antibiotic therapy is paramount. Data on the use of aPC in severe malaria or severe leptospirosis needs to be generated urgently but the role of syndromic evaluation and empiric therapy must not be relegated.


In view of the fact that a significant proportion of neonates with malaria may be missed on our wards on the assumption that the disease condition is rare, this study aims at documenting the prevalence of malaria in neonates admitted into our neonatal ward. Specifically, we hope to describe its clinical features and outcome of this illness. Knowledge of these may ensure early diagnosis and institution of prompt management.st December 1999 were reviewed. All neonates (fifty-seven) who had a positive blood smear for the malaria parasite were included in the study. Socio-demographic data as well as clinical correlates of each of the patients were reviewed. The Epi-Info 6 statistical software was used for data entry, validation and analysis. A frequency distribution was generated for categorical variables. To test for an association between categorical variables, the chi-square test was used. The level of significance was put at values less than 5%.Methods Hospital records of all patients (two hundred and thirty) admitted into the Neonatal ward of Olabisi Onabanjo University Teaching Hospital, Sagamu between 1st January 1998 and 31Prevalence of neonatal malaria in this study was 24.8% and 17.4% for congenital malaria.While the mean duration of illness was 3.60 days, it varied from 5.14 days in those that died and and 3.55 in those that survived respectively. The duration of illness significantly affected the outcome . Fever alone was the clinical presentation in 44 (77.4%) of the patients. Maturity of the baby, sex and age did not significantly affect infestation. However, history of malaria/febrile illness within the 2 weeks preceding the delivery was present in 61.2% of the mothers. Maternal age, concurrent infection and duration of illness all significantly affected the outcome of illness. Forty-two (73.7%) of the babies were discharged home in satisfactory condition.It was concluded that taking a blood smear to check for the presence of the malaria parasite should be included as part of routine workup for all neonates with fever or those whose mothers have history of fever two weeks prior to delivery. In addition, health education of pregnant mothers in the antenatal clinic should include early care-seeking for newborns. Malaria has been recognised as a leading cause of infant morbidity and mortality in Africans  but neonAs clinical signs of neonatal malaria may be indistinguishable from that of neonatal sepsis, it has been suggested that screening for malaria parasite be included as part of routine investigation in newborn infants with fever . SometimThe burden of malaria in non-malarious countries had led to the suggestion by workers that neonatal malaria is to be considered in those newborns with congenital infection born to mothers who had travelled to endemic areas, even when they appear clinically healthy ,7.A cursory observation on our neonatal ward suggests that malaria may not be uncommon in this environment. However, lack of scientific documentation has hindered the development of a definite policy on case management of neonatal malaria in our hospital.Hence, this study aims at documenting the prevalence of diagnosed malaria in neonates admitted into our neonatal ward and description of its clinical features. In addition, we sought to determine the contributory factors to the outcome of the illness.This we hope may ensure early diagnosis and institution of prompt management.st January 1998 and 31st December 1999 were reviewed. This is the Teaching Hospital of the Olabisi Onabanjo University. The bed capacity of the neonatal ward is twenty-six (26).This is a retrospective descriptive study. Case notes of all patients admitted into the Neonatal ward of Olabisi Onabanjo University Teaching Hospital, Sagamu, Ogun State, Western Nigeria between 1Majority of the babies admitted into the ward are referred from the nearby general hospital and private hospitals within and outside the state. The only routine investigation in the unit is a sepsis work-up that is usually done in the service laboratory of the teaching Hospital. . All neonates whose record indicate positive blood smear for malaria parasite were included in this study. (no record of density of parasitemia was available).Other data extracted from such baby's record included: age at onset of symptoms (days), sex, birth weight, length, place of delivery, presenting problem that immediately led to blood investigation for malaria parasite and duration of illness, assessed or known gestational age, as well as treatment regimen given and outcome of illness. Results of laboratory investigations viz: blood culture, full blood count and erythrocyte sedimentation rate were reviewed as well. The mothers' age, parity, history of febrile illness/malaria at least 2 weeks preceding the delivery and educational level were retrieved from the case notes.For the purpose of the study, Neonatal malaria is defined as symptoms attributable only to malaria with evidence of ring forms of malaria parasite in the erythrocyte of an infant within the first twenty-eight days of life. Congenital malaria is defined as symptoms attributable only to malaria with evidence of ring forms of malaria parasite in the erythrocytes of an infant within the first seven days of life.The Epi-Info 6 statistical software was used for data entry, validation and analysis. Frequency distribution was generated for categorical variables. To test for association between categorical variables, the chi-square test was used. Level of significance was put at values less than 5%.Fifty-seven (24.8%) of the two hundred and thirty admissions into the neonatal ward in the period under review had a positive blood smear for malaria parasite. Eighty-nine out of the two hundred and thirty admissions were screened for malaria (38.7%).Those with positive blood smears were aged 1–28 days. Forty (70.2%) of the babies were aged 7 days or less and twenty- five (43.3%) were less than 48 hours old at the time of presentation. The mean age was 6.7 days. There were 35 males and 22 females giving a male: female ratio of 1.6: 1. Infestation is not sex-linked .Almost forty- four percent (43.9%) of the babies weighed less than 2.5 kg and 56.1% were more than 2.5 kg at the time of presentation. Mean weight was 2.58 kg. Length of 73.2 % of the babies was less than 48 cm. Mean length was 44.29 (SD 4.33).)As shown in table As shown in table Twenty- eight of the babies were delivered in public hospitals while 21 were delivered in private hospitals and the others were delivered elsewhere. The place of delivery did not significantly affect infestation. While 21 of the babies were pre-term, 25 were full-term and maturity was not documented in eleven (11) of the babies. Forty- two (42) of the babies were however appropriate for age and 10 were small for date. There was no significant difference in the rate of infestation between pre-term and full-term babies .Three of the mothers were aged between 15–20 years, Seven, between 21 and 25 years and 14 were between 26–35 years. Maternal age was not documented in 23 cases. Maternal educational level was documented in only six cases. Febrile illness within the last trimester of the pregnancy was present in 30 out of the 40 cases in whom there are records. No information was however recorded for 17 cases. Infestation is significantly higher in babies of mothers with history of febrile illness .Only 27 of the mothers were primipara, 20 were multipara, 3 Grand multipara. The parity of 7 of the mothers was not stated.No hematological evidence of bacterial infection was found in 96.4% of the babies and thirty – seven (64.9%) had Packed Cell Volume (PCV) between 30–45%. None of them had Packed Cell Volume (PCV) less than 20%.Table Fifty of the babies were treated with appropriate doses of chloroquine (25 mg chloroquine base/kg), one had Sulfadoxine-Pyrimethamine (SP). No treatment was documented in 6 cases. Forty-two of the babies responded to the antimalarial given and were subsequently discharged home in satisfactory condition. Four died and 11 were discharged against medical advice.The duration of fever in those that died was longer than 5 days. This significantly affected the outcome of illness . Also, concurrent bacterial infection and maternal age significantly affected the outcome of illness . Three out of the 4 deaths occurred in the babies of mothers aged 15–20 years. Apart from history of febrile illness in the mother within two weeks prior to delivery, none of the other factors examined is significantly associated with infestation.This study has identified a prevalence of 24.8 % for diagnosed neonatal malaria and 17.4% for congenital malaria. This is in keeping with an African survey on congenital malaria that had reported values of 0–23% [Similar to our findings in this study, a report from western Uganda has documented that there is no association between congenital parasitemia and birth weight . AlthougHowever, that history of febrile illness in the last trimester of the pregnancy is significantly associated with infestation seem to suggest that all babies of such mothers with such history should be placed under observation in the neonatal ward and screened for malaria parasite.The parasitology report of the six babies whose record of treatment could not be traced was probably retrieved after their exit from the hospital. They were part of the eleven that discharged against medical advice. Interestingly, they gave dissatisfaction with clinical progress of their babies as reasons for discharge. They had been on admission for some-time and the blood smear was only requested for when it appeared that the infants' condition was not improving.Maternal age, concurrent bacterial infection and duration of illness significantly affected the outcome of illness. The three are interrelated. Babies of teenage mothers are at risk of neonatal infection. Such mothers may also present late in the hospital either due to poverty or ignorance.In conclusion, this study has further confirmed that neonatal malaria is not as rare as we had thought in Sub-Saharan Africa. Clinicians need to have a high index of suspicion so that rapid diagnosis and early appropriate intervention can be instituted in line with the concept of Roll Back Malaria.1. All neonates with features suggestive of neonatal infection should have blood smear for malaria parasite as part of their routine screening.2. All babies of mothers with history of febrile illness within the last two weeks of pregnancy should have blood smear for malaria parasite examination.3. Early care seeking for neonates should be part of the educational talks in the antenatal clinics.The author(s) declare that they have no competing interests.TIR had the concept and wrote the article, OO analysed the data and MF entered the data. The three authors read through and agreed on the content of the article.The pre-publication history for this paper can be accessed here:


Although maternal anaemia often stems from malaria infection during pregnancy, its effects on foetal haemoglobin levels are not straightforward. Lower-than-expected cord haemoglobin values in malarious versus non-malarious regions were noted by one review, which hypothesized they resulted from foetal immune activation to maternal malaria. This study addressed this idea by examining cord haemoglobin levels in relation to maternal malaria, anaemia, and markers of foetal immune activation.Cord haemoglobin levels were examined in 32 malaria-infected and 58 uninfected women in Blantyre, Malawi, in relation to maternal haemoglobin levels, malaria status, and markers of foetal haematological status, hypoxia, and inflammation, including TNF-α, TGF-β, and ferritin. All women were HIV-negative.Although malaria was associated with a reduction in maternal haemoglobin , no reduction in cord haemoglobin and no significant relationship between maternal and cord haemoglobin levels were found. Cord blood markers of haematological and hypoxic statuses did not differ between malaria-infected and uninfected women. Maternal malaria was associated with decreased TGF-β and increased cord ferritin, the latter of which was positively correlated with parasitaemia . Increased cord ferritin was associated with significantly decreased birth weight and gestational length, although maternal and cord haemoglobin levels and malaria status had no effect on birth outcome.In this population, cord haemoglobin levels were protected from the effect of maternal malaria. However, decreased TGF-β and elevated ferritin levels in cord blood suggest foetal immune activation to maternal malaria, which may help explain poor birth outcomes. Malaria affects more than three million pregnant women per year in developing countries, where it commonly causes poor birth outcomes and maternal anaemia -3. BrabiIn non-malarious regions, maternal anaemia may stem chiefly from iron deficiency; higher cord haemoglobin levels may thus reflect a response to foetal hypoxia caused by decreased placental oxygen transport . In malaTo further address the hypothesis that cord haemoglobin levels in malarious areas are lower than expected due to foetal immune activation to maternal malaria, cord haemoglobin levels were examined in this study in malaria-infected and uninfected women in relation to maternal malaria status and haemoglobin levels and markers of foetal haematological, inflammatory, and hypoxic status. In specific, chronic foetal hypoxia was assessed via levels of erythropoietin (Epo) , a hormoAs markers of interest were measured both in maternal peripheral and umbilical cord blood, one critical issue is whether the cord measures truly reflect foetal production rather than placental transfer of maternally produced factors. Human data addressing this topic is limited, given the invasive and/or complex nature of the required studies. In general, studies suggest that most substances can cross the placenta, with the rate dependent on molecular weight, such that those with larger molecular weights cross slowly and those with molecular weights under 500 daltons quite rapidly . ErythroNinety pregnant women pre-labor or in the latent phase of labor attending the Labour Ward, Queen Elizabeth Central Hospital, Blantyre, Malawi, were recruited from February to October 2002 as part of a prospective cohort study investigating the impact of maternal malaria on HIV vertical transmission . All womMaternal socio-economic , health  and anthropometric ) data were collected. Neonatal anthropometrics  were evaluated within the first 24 hours after birth. Gestational age was determined by the New Ballard Score .Maternal peripheral blood samples were taken at recruitment, and cord and placental samples were collected at delivery. Thick blood films were prepared from these samples for malaria microscopy. Placental blood was collected into EDTA by incising the cleaned maternal surface of the placenta and aspirating blood welling from the incision with a sterile pipette. Samples were separated within one hour, and plasma was stored at -70°C. Placental biopsies from a pericentric area  were placed into 10% neutral buffered formalin to be used for placental malaria histopathology.Plasmodium falciparum malaria parasitaemia per μl was determined by counting parasites per 200 leukocytes, assuming 6000 leukocytes per μl [Thick blood films were air-dried and Field's stained. s per μl . Placents per μl ; stage os per μl . For theAll subjects were consented and received HIV pre-test and post-test counseling before the onset of active labor. HIV status was determined by Serocard rapid test for HIV-1 and 2  and Determine HIV1/2 ; disagreement between tests was settled by HIV-SPOT ELISA .® haemoglobinometer ; some results were not available as samples clotted. Maternal peripheral and cord plasma soluble transferrin receptor (sTfR) and ferritin were assayed by enzyme-linked immunosorbent assay (ELISA) kits according to manufacturer's instructions . Data were analysed in Microsoft Excel using a log-log standard curve. Limits of detection were as follows: 1 μg/ml (sTfR) and 0.59 ng/ml (ferritin).Haemoglobin (Hb) concentration were measured in peripheral and cord blood samples using a HemocueMaternal peripheral, placental and cord plasma tumor necrosis factor-α (TNF-α) and transforming growth factor-β (TGF-β) and maternal peripheral and cord plasma C reactive protein (CRP) levels were also assayed by ELISA . Color was developed by SigmaFast o-phenylenediamine dihydrochloride tablets . Data were analysed as described above. Limits of detection were as follows: 4 pg/ml (TNF-α), 13 pg/ml (TGF-β), and 1 μg/ml (CRP).Because scalp blood gas analysis, the most accurate means of assessing foetal hypoxia, was not feasible in the study setting, other proxies for chronic foetal hypoxia were measured: erythropoietin (Epo) , and corThis study was approved by the College of Medicine Research Committee, University of Malawi, and the Institutional Review Boards of the University of Michigan and the University of North Carolina.Data were entered in MS-Access and analyses were performed using SPSS v.10 and Intercooled Stata 8.0. Skewed data were log or (log+1) transformed before certain analyses. Maternal characteristics in malaria-infected and uninfected women Table  were comOf the 90 study participants, 29 had maternal peripheral malaria infections, 32 had placental malaria infections, and 42 had infections in one or both compartments. No cord blood malaria was identified in this study. Table Table Data on the effects of malaria on maternal and foetal markers of haematological status, inflammation, and chronic hypoxia are presented in two tables: Table The effect of malaria on haematological status differed markedly between women and neonates. Compared to their uninfected counterparts, women characterized by any of the three definitions of malaria infection tended to have significantly lower maternal Hb concentrations . Unsurprisingly, therefore, neither decreased maternal nor cord Hb was significantly associated with poorer birth outcomes in malaria-infected women (data not shown), although cord sTfR levels, which were not different between neonates of malaria-infected and uninfected women Tables  and 4, wIn this study, the haematological status of neonates of mothers with malaria parasitaemia at delivery was not significantly impacted. Despite the effect of malaria on maternal Hb, which was expectedly lower, neonatal Hb levels were not decreased in the cord blood of malaria-infected compared to uninfected women. In addition, cord Hb values were not correlated to maternal Hb concentration in either group or severity of malaria, as described in the Results section. Although the sample size was relatively small, these results resemble more closely the results from Western studies, which tend to find unaffected cord values, than the studies of malaria- and non-malaria-exposed mother-neonate pairs in developing countries, as reviewed by Brabin . It is uFerritin is an iron storage protein that is an acute phase reactant in the presence of infection. While cord ferritin decreases in foetuses of anaemic mothers without malaria, it appears to increase in foetuses of mothers with increasing parasitaemia. Increased ferritin in the presence of inflammation has been suggested to reflect an evolutionary strategy by which the body removes bioavailable iron from the presence of parasites . In foetMany researchers have assumed that foetal hypoxia, rather than inflammation, mediates the effect of malaria and maternal anaemia on birth outcomes, particularly low birth weight. Despite the induction of foetal hypoxia by maternal anaemia ,33 and tAllen  hypothesIn summary, cord haemoglobin levels were unchanged by maternal malaria and anaemia during pregnancy in this population. However, maternal parasitaemia induced a foetal inflammatory response, specifically lower TGF-β and higher ferritin levels. Elevated cord ferritin in turn was associated with lower birthweights and shorter gestations.ETA was responsible for conception and design of the study; IRB approval; conducting assays; analysis and interpretation of data; drafting the paper; and revising it for publication. JK and DDK performed many of the assays and analyses of these assays. VM designed the larger study and conducted much of the data analysis of that study that formed the foundation of this one. ET and VLM supervised patient enrollment and acquisition of data and aided in its submission to the IRB. MEM and SRM were central to the conception and design of the study, the analysis and interpretation of data, and the revision of the paper for publication. SRM was involved in the conception and design of the study; IRB approval; analysis and interpretation of data; drafting the paper; and revising the paper for publication.


Very little is known about the management of malaria and treatment-seeking patterns among children and adults in areas of seasonal malaria transmission particularly in east Africa.The aim of this study was to assess treatment-seeking behaviour for reported malaria among all age groups in an area of seasonal transmission.kebeles in Adami Tulu district in south-central Ethiopia, during October-November 2003, using a pre-tested interviewer-administered structured questionnaire.A community-based cross-sectional study was carried out among 2,253 households in 12 randomly selected rural Reported malaria was 14% among 12,225 people assessed during the last 14 days. Family/self-diagnosis was most common and the main first responses included visiting village-based community health workers (CHWs) (33%), public health facility (23%) and private clinic (17%). Home treatment was the least reported first response (3%). Only 13% had sought treatment within the first 24 hours of symptom onset. Early treatment-seeking pattern was reported among those who visited CHWs and practiced home treatment, with more delays among public facility users. Treatment-seeking behaviour was similar in all age groups.A considerable proportion of visits were made to CHWs and private providers, necessitating the importance of strengthening both community-based interventions and peripheral public and private facilities. Finally, the community should be informed and educated about the importance of early diagnosis and prompt treatment with effective antimalarials. Malaria remains the major cause of morbidity and mortality particularly in sub-Saharan Africa ,2. In arIn the past, treatment-seeking behaviours have been extensively studied in areas of high endemicity ,8. StudiDespite the extensive work on treatment-seeking behaviours for malaria particularly among children under 5 years of age in areas of intense transmission in sub-Saharan Africa, little is known about the management of malaria and treatment-seeking patterns among children and adults in areas of seasonal malaria transmission ,14-16. D2. Rainfall is inadequate and seasonal, and lasts between the months of May and November with the greatest intensity during June-August. In 2002, a projected population of the district (from 1994 census) was 145,000 people with a density of 104/km2. The population is predominantly subsistence farmers belonging to the Oromo ethnic group and Muslim denomination. Most of the households particularly in rural areas are poor and the majority of the houses are circular tukuls with thatched conical roofs.This community-based cross-sectional study was conducted during October-November 2003 in the rural areas of Adami Tulu district in south-central Ethiopia. The capital of the district, Zeway, is located at about 160 kms south-east of Addis Ababa. The district generally lies at an altitude between 1,500 and 1,600 m above sea level and covers an area of 1,403 kmPlasmodium falciparum and Plasmodium vivax constituted 66% and 31% of the infections, respectively [Malaria transmission in the district is seasonal, with the major transmission from September to December; following a heavy summer rainfall . The stuectively .P. falciparum on the 14-days follow-up, much higher than the national mean treatment failure rate of 36% [®) as the first-line treatment for uncomplicated falciparum malaria [Anopheles arabiensis, and Anopheles pharoensis is considered as a secondary vector [Chloroquine and sulfadoxine-pyrimethamine (SP) resistant falciparum malaria is widespread in the district. In the therapeutic study of SP during October-December 2003 in the district, a total treatment failure rate of 53.4% and late parasitological failure rate of 43.1% were reported against e of 36% . Despitee of 36% . In 2004 malaria . Most may vector .The district had two health centers, three health stations, one malaria control laboratory (MCL) center, three health posts, 15 medium and lower private clinics, 34 community-health workers (CHWs) specifically trained for village-based malaria treatment, one missionary clinic, six drug shops and seven rural drug vendors. Most of these health care facilities were located in Zeway and three other small towns. There was no hospital in the district. As is true in many areas, there could also be a number of unregistered drug outlets in the district.kebeles and four towns. Kebele is the lowest administrative unit with a population of 1,000 to 3,000. A list of all rural kebeles, including the estimated number of households and population, was obtained. Based on geographical homogeneity and history of malaria problem, all kebeles in the district were classified into three strata with the assistance of the District Health Office (DHO) malaria control experts. Finally, four kebeles were randomly selected per stratum; thus, making a total of 12 kebeles for the study. All households in the selected kebeles were included in the survey through house-to-house visits. Household was defined as a group of people including husband, wife, children or others living together sharing the same house.The district is administratively organized into 62 rural kebeles and trained for five days collected the data using the local language . In the study area, malaria is locally called "busa". The author and two health workers from the DHO supervised the data collection.In each household, the head (or the spouse), or a representative was interviewed using a pre-tested interviewer-administered structured questionnaire about treatment-seeking behaviour for self-reported malaria illness two weeks prior to the interview. The respondents were asked to list all members of their households, and each respondent was asked to think about the last 14 days and indicate any one of the household members who encountered malaria episode. Information was specifically obtained on the symptoms, sources, timing and treatment of reported malaria for all age groups. In order to help the respondent recall the event and practices of seeking treatment, other adult members of the household or sick adults were also participated in answering the questions. For children with reported malaria illness, either the head of household or the mother (or caretaker) or both of them were participated in answering the questions. Twelve local interviewers recruited from the selected X2) test and a P-value of < 0.05 was considered significant. The health care providers were classified as (1) public health facilities such as health post, health station, health center, MCL and hospital; (2) private clinic that included high or medium level clinics; (3) CHWs rendering malaria treatment at village level; and (4) home treatment practices. The ages of the reported malaria patients were categorized into children (<5 years and 5–14 years) and adults (≥15 years) for comparison.Data were entered and analysed using Epi Info software version 6.04d . Frequencies and proportions were used for the descriptive analysis. Differences in proportions were compared for significance using Chi-square . Verbal informed consent was obtained from all respondents who participated in the study after explaining the purpose and objectives of the study. Any patient with symptoms suggestive of clinical malaria was treated by SP according to the national guidelines at the time of the study . Patientkebeles, data were collected from 2,195 (97%) households. Malaria illness was reported for 14% (n = 1748) of individuals among 12,225 people assessed during 14 days prior to the interview, 70% (n = 1222) of which had recovered on the day of interview. The mean (± SD) age of the patients was 19 (± 15.7) years. The majority of the patients were adults ≥20 years (40%), followed by children <10 year-old (36%). There were about 50% of female patients. The majorities of the reported malaria cases aged ten or above were married (57%), illiterate (71.3%), farmers/housewives (61%) and students (32%).Of 2,253 visited households in 12 rural P = 0.013). However, vomiting was more reported among <5 children compared to children between 5–14 year-old and adults ≥15 .In all age groups of patients, the most commonly reported symptoms were fever, shivering and chills, loss of appetite, headache and high body temperature Table . ReporteThe two-week prevalence of reported malaria across the three age group categories indicated that adults' prevalence was higher than that reported for children Table . At the Table Among those who sought any type of treatment, only 13% had sought treatment within the first 24 hours of onset of illness  declare that they have no competing interests.WD was the principal investigator of all aspects of the study from conception and design to the final analysis and preparation of the final manuscript. He is also the guarantor of the paper.


The study sought to determine the effect of using insecticide-treated clothes (ITCs) on personal protection against malaria infection. The specific objectives were to determine the effect of using ITCs on the rate of infection with malaria parasites and the effect on indoor mosquito density.Diras, Saris, Jalbaabs, Ma'awis and shirts) and their bedding (sheets and blankets) treated with insecticide (permethrin). The other half (comparison group) had their clothes treated with placebo (plain water). Indoor mosquito density was determined from twelve households belonging to the participants; six in the treatment block and six in the comparison block.  During pre-test and post-test, laboratory analysis of blood samples was done, indoor mosquito density determined and questionnaires administered. Using STATA statistical package, tests for significant difference between the two groups were conducted.This study was done in Dadaab refugee camps, North Eastern Province Kenya between April and August 2002, and involved a total of 198 participants, all refugees of Somali origin. The participants were selected through multi-stage cluster sampling. Half of the participants (treatment group) had their personal clothes worn on a daily basis (Use of ITCs reduced both malaria infection rates and indoor mosquito density significantly. The odds of malaria infection in the intervention group were reduced by about 70 percent. The idea of using ITCs for malaria infection control was easily accepted among the refugees and they considered it beneficial. No side effects related to use of the ITCs were observed from the participants.The use of ITCs reduces malaria infection rate and has potential as an appropriate method of malaria control. It is recommended, therefore, that this strategy be considered for use among poor communities like slum dwellers and other underprivileged communities, such as street children and refugees, especially during an influx to malaria-prone regions. Further research on cost-effectiveness and sustainability of this strategy is worthwhile. Malaria is the world's most prevalent vector borne disease. Each year 300 to 500 million clinical cases of malaria occur and at least 1 million people die of malaria each year .The poor bear the highest burden of malaria: they are at a higher risk of becoming infected with malaria, because they live in dwellings that offer little protection from mosquitoes yet they may not afford protection methods like insecticide-treated nets (ITNs) . ChildreThe WHO "Action Plan for the reduction of reliance on DDT in Disease Vector Control" recommends research into "the effectiveness, sustainability, and affordability" of insecticide-treated materials as one of the handful of approaches to replacing DDT use in malaria vector control . ITNs haAccording to Kenya Demographic Survey (KDHS) 2003, despite the fact that two third of Kenyans live in malaria-endemic areas, only 22 percent of households in Kenya have at least one mosquito net, while only six percent have at least one ITN. Only 10 percent have more than one net, while three percent have more than one ITN, despite the fact that an average Kenyan household size is 4.4 persons . StudiesThese problems associated with use of ITNs limit their use especially in poor communities and other underprivileged communities like refugees. This points out to the need to utilize less expensive alternatives and more appropriate malaria control strategies in such communities. One potential method for malaria vector control that may meet the needs of these groups is insecticide-treated clothes (ITCs). However little research and consequently social marketing of the strategy has been done.Results of a randomized controlled study done in Afghan refugee camp in 1996 showed potential of ITCs in reducing malaria infection for people aged less than 20 years . The curThe study was undertaken between April and August 2002 in Dadaab refugee camps, Garissa district, Kenya. In Dadaab refugee camps, mosquitoes breed mainly in pools formed by tap stand spillage. Other breeding sites included borrow pits, water pools formed during rains and animal watering cans.There are three sub refugee camps in Dadaab; Ifo, Hagadera and Dagahaley. All the sub camps are divided into sections, which are further sub-divided into blocks. An average of nine blocks makes a section. Each block consists of about 600–700 people and consists of several households  each consisting of an average of six people. The refugee population in the three camps is about 130,000 people; about 97 percent are from Somalia, while about three percent are Ethiopians, Sudanese, Ugandans and Eritreans. The study was undertaken in the Ifo sub-camp with a total population of about 45,000 people.dira  and a long dress known as jalbaab to cover the head and the rest of the body respectively. Similarly, the men wear a long skirt-like wrap known as ma'awis.The Somali refugees are predominantly Muslim and wear long clothes; the women wear a veil or wrap known as Many of the refugees have no source of income and rely almost entirely on assistance from aid agencies operating in the area including UNHCR, WFP, Care International, MSF-Belgium, and GTZ. They mainly live in makeshift houses some made up of earth and others of polythene paper, old clothes or sacks.This was a community trial which had a treatment arm and a comparison arm. Pre-test was done at the beginning of the study, then the intervention was given, the participants were followed up for about three months and post-test was done.The study sample included refugees  of Somali origin of all ages who did not have any plans of moving from the camps for the six months following the initiation of the study and had no known severe skin disease, known allergic reactions to chemicals or severe respiratory problem.Multistage cluster sampling was employed to select a sample of 198 participants; at the first stage, Ifo sub-camp was selected from the three sub-camps purposely because it was reported to have higher morbidity from malaria, at the second stage, two sections from Ifo sub-camp were selected at a distance of about one and a half kilometers apart (to cater for mosquito flight range). At the third stage, one block from each of these sections were selected randomly; one block was assigned treatment and the other comparison block randomly.At the fourth stage, from the two blocks, 30 households (14 in the treatment block and 16 in the comparison block) were selected by systematic random sampling from a total of about 200 households; 100 in each block (therefore about 15 percent of the households in each block participated in the study). All the individuals residing in the selected household were recruited into the study. A total of 198 participants, 97 in the treatment and 101 in the comparison block were recruited.Those in the treatment group had their clothes treated with insecticide while the comparison group had their materials treated with a placebo (plain water). The treatment of clothes was done by the lead researcher assisted by field assistants who were trained before the start of the intervention. The research participants were not allowed to handle the chemical and the chemical was also not being sold in the local market thus chances of cross-intervention were remote. In addition, treatment being done to personal clothes made any possibility of giving out treated materials to those in the comparison group remote, the effect of which would create some level of bias.Double blinding was done for malaria parasite smear in that the participants did not know whether they were in the treatment or the comparison group and the laboratory technologist  was also not told which slides belonged to the treatment or comparison group.Gudomeyas) on the study and then through their assistance, the community was mobilized. This was done in the second week before intervention was done.Training of field assistants was done in the first week of the study before the intervention. The field assistants were recruited from among Care Community Health Workers and MSF trained auxiliary nurses. After the training of field staff, with their assistance, community mobilization was done by first briefing the community leaders . This was done to clear the participants of malaria parasites before the start of the study.diras, saris, jalbaabs, ma'awis, and shirts) and beddings (sheets and blankets) with insecticide was done for the treatment group. Permethrin (Peripel EC 55) manufactured by AgrEvo at a concentration of 15 mls/4000 mls of water was used for treating the fabrics through dipping. The treatment was repeated every after three weeks during the study period. The treatment was repeated following the recommendations by the manufacturers of the insecticide that re-treatment should be done every after about five washes and the community indicated they washed personal clothes about twice a week. The comparison group had their materials dipped in plain water (acting as a placebo) at same intervals.Treatment of personal clothes . The first post intervention test was done about two weeks after the treatment of clothes . The samples were taken by field staff who had been trained as auxiliary nurses by MSF. The blood samples were stained with Giemsa and analysed using a light microscope. The laboratory analysis was done by a trained lab technologist from MSF at the MSF laboratory. The laboratory analyst was blinded on which slides belonged to the intervention or control group to reduce bias.For those whose blood samples were found to have malaria parasites, they were taken to the MSF health facility and were treated with anti-malarial drugs (sulfadoxine-pyrimethamine). For blood samples, participants were tracked for three days after which they were regarded as absent. Thus, during the second and third post intervention period, some of the participants could not be traced. Most of those missing were reported to have gone on vacation to a different sub-camp.Indoor mosquito density was also measured twice after intervention, about one week after blood samples were taken. Mosquitoes were collected from the houses at around six to seven in the morning using pyrethrum spray catch method. Each household was visited once per period. The collection of mosquitoes was done by field staff from vector and pest control unit in Care-Refugee assistance project (Care-RAP). Analysis of the mosquitoes was done with assistance of an entomologist from the vector and pest control unit, Care-RAP. Mouthparts were used to identify the species and sex of the mosquitoes. The status of the mosquito, whether engorged or not was established. Identification was done by help of a dissecting microscope. Participants were observed during the follow-up period for side effects including reactions to the skin, effect on the respiratory system or any complaints/symptoms. A simple survey was done at the end of the study through interviewing households to determine the experiences in using the treated materials and rates of self reported malaria.Using the STATA statistical package, analysis involving comparing the two samples (treatment and comparison) was done. Odds Ratio (OR) was calculated to determine difference between the treatment and the comparison group. Poisson regression was done for mosquito density while logistic regression analysis was done for malaria infection status. For the logistic regression, various factors that could be associated with malaria infection were controlled for in the regression models. Cases with at least one record of information on the dependent variable  during the follow-up period after intervention were considered. If there was no data collected on cases regarding their malaria infection status during the two data collection periods following intervention, the cases were deleted . For all the tests statistical significance was assumed at 5 percent level.The study was approved by the Moi University institutional ethical clearance board before it was undertaken. The fundamental principles of ethics in research on human participants were upheld throughout the project. The research procedures were disclosed to the participants and informed consent was sought from the participants through a signed consent form. Nobody was coerced into the study and if one wished to withdraw, they were allowed to do so without prejudice; two households actually withdrew from the study. This study was supported by Care International Refugee Assistance Project and there was plan that should the strategy be found to be effective, Care would implement the strategy in the refugee camps thus the comparison group would have their clothes treated with the insecticide.A total of 198 participants from 30 households were admitted into the study; 97 participants in the treatment group from 14 households and 101 in the comparison group from 16 households. Blood samples for malaria parasite smear were taken from 193 participants; 96 from treatment group and 97 from the comparison group. Mosquito density was assessed from 6 households in each group. As seen from Table During the post-intervention period, the number of mosquitoes collected from the treatment block decreased compared to the number collected during pre-intervention period. In contrast, the number of mosquitoes collected from the comparison group increased compared to the number collected during pre-intervention. Reduction of mosquito density in the intervention households was significant for total mosquito density, engorged mosquitoes , female mosquitoes and anopheles mosquitoes but was not significant for female anopheles mosquitoes Table .During the post-intervention period, malaria parasite test was done twice. The status was indicated as positive if during any of the tests the participant had a smear-positive slide and negative if the two tests were negative for the individual Table . Blood sA logistic regression was done with malaria infection status as the dependent variable to control for various factors that could affect malaria infection rate Table . ClusterIn the third model, several characteristics including possession of nets, sex, age, use of other malaria control methods and sleeping outside were added to the model. These factors may not be directly related to malaria infection but they can alter the risk of infection with malaria parasites. When all these factors were controlled, the odds Ratio changed to 0.29 (P < 0.001). This indicates that the odds of being infected with malaria when all these factors were controlled were reduced by 71 percent.At the on-set of the study, the community was very enthusiastic about the study and everyone selected accepted to be part of the study. However, during the last post-intervention data collection, one household from the comparison group withdrew completely from the study saying the study had no benefits to them. This household refused to have blood samples taken from the members during the last post-intervention period and also to have mosquitoes collected from their household in the last post intervention data collection period. This household was replaced with the immediate neighbouring house for mosquito density, but was not replaced for malaria infection status. Another household from the comparison group refused to have blood samples taken from them during the last post intervention period but allowed mosquitoes to be collected.The information on perceptions of malaria morbidity was collected during the last month of the study. Participants were asked whether any member of the family had suffered from malaria during the past one month. About nine percent of the participants were reported to have had malaria during the past one month. More people in the comparison group (13 percent) were reported to have had malaria than in the treatment group (5 percent) and the difference was statistically significant .Observation for side effects was done during the follow-up period. No side effects were observed among the participants or reported by the participants from the use of the treatment throughout the study period. All the participants in the treatment group said the treatment was advantageous. Among the advantages given were that use of the treated clothes reduced the mosquitoes in the house and mosquito bites, and that other insects like bedbugs and body/head lice in children were reduced. They also said that children no longer had problems with sleep at night because there was no nuisance from mosquito bites; that children hardly woke-up in the middle of the night and slept until 8 am in the morning nonstop, as opposed to earlier times when they used to wake up at night crying.Those in the comparison group said they perceived no benefit from the study apart from two households who said mosquitoes were reduced but only for the first one night after intervention. One family from the comparison group reported that they thought that the treatment was attracting mosquitoes to their household. This is the household that withdrew completely from the study.shukas) in the pastoral community in Kenya [This study shows that insecticide-treated clothes are protective against malaria. The odds of being infected with malaria for those in the treatment group were significantly reduced when all ages were combined. When the odds were calculated by age groups, the odds were significantly reduced for older children (age 5–14years), youth age 15–24years) and the older people (50 years and above). For children less than five years and the adults 25–49 yeara, the results showed no benefit. This study adds to the body of knowledge on the potential for ITCs or ITMs as an alternative to ITNs. A similar study of relevance is a trial of permethrin-impregnated bedsheets . This explains why there were very many culicine mosquitoes and very few Anopheles mosquitoes among the mosquitoes collected in the houses. The density of mosquitoes collected from the treatment block was less than that of the comparison block.In the current study, despite the fact that the region where Dadaab refugee camps are located has low risk of malaria generally, the risk of malaria infection at the camps may be higher than in the surrounding areas because of poor sanitary practices at the camps. The refugees in Dadaab fetch water from communal taps located at several positions in the camps. Water spilled at the tap-stands contributes a lot in breeding of mosquitoes in the refugee camps. The overall percentage of those infected with malaria parasites before the intervention was low compared to the months after the intervention. This could be explained by the season preceding the start of the study, which was quite dry. By the time the first post-intervention data was collected, the rains had somehow stopped but there were water pools still lying about. During this period, the indoor mosquito densities in the comparison group increased as compared to the pre-intervention densities. This could be due to weather changes: mosquitoes bred in the water pools formed by the rains because though the rains had stopped, the flat terrain and poor drainage of the area encouraged persistence of the water pools and, thus, mosquito breeding. If nature was left to take its own course, the same would have been expected to happen in the comparison group. However, the densities in the treatment group decreased as compared to the pre-intervention period. This could be explained by the intervention given to this group because as expected with permethrin, most mosquitoes coming to the house were either repelled by the ITCs or were knocked down. Accordingly, there were fewer blood-fed mosquitoes in the treatment group as compared to the comparison group (though difference not significant). For the comparison group, this could be explained by the fact that mosquito breeding had increased following the rains. During the second post-intervention data collection, the rains had stopped and the water pools formed by the rains had somehow dried up. Most breeding of mosquitoes was happening at the tap-stand spillage and at pit latrines  was similar to that of treating bed nets and cost only 10–20 percent of the price of a new bed net . This poNotwithstanding the findings on the potentiality of ITCs in malaria infection control in this study, further research requires to be done to assess cost-effectiveness and sustainability of this strategy.It was concluded that use of ITCs reduces mosquito densities in the house and the rate of infection with malaria parasites. The reduction in the nuisance caused by mosquito bites cannot be overemphasized. It is recommended that this strategy be considered for use in the control of malaria and nuisance caused by mosquito bites in vulnerable communities like poor communities living in slum areas as poor people are less likely to afford ITNs, and if they do, they may not afford enough ITNS to cater for the whole household and in such a circumstance, ITCs could complement the ITNs. The strategy is also recommended for refugee communities especially during emergencies like during influx of refugees to a malaria-prone region as some refugees may not have immunity to malaria. However, a larger scale study for a longer period to traverse different seasons and to determine sustainability and possibly cost-effectiveness of the strategy in a community set up is worthwhile.KEW conceived and designed the study, collected and analysed the data, drafted the manuscript and gave final approval for publishing. VJM contributed to the design of the study, supervised data collection, reviewed the manuscript and gave final approval for publishing. KIW contributed to the design of the study, participated in data collection, reviewed the manuscript and gave final approval for publishing. MF contributed to the analysis of data, reviewed the manuscript and gave final approval for publishing.