Countries aiming for malaria elimination require a detailed understanding of the current intensity of malaria transmission within their national borders. National household sample surveys are now being used to define infection prevalence but these are less efficient in areas of exceptionally low endemicity. Here we present the results of a national malaria indicator survey in the Republic of Djibouti, the first in sub-Saharan Africa to combine parasitological and serological markers of malaria, to evaluate the extent of transmission in the country and explore the potential for elimination.19) of Plasmodium falciparum exposure. Multivariate regression analysis was used to determine the risk factors for P. falciparum infection and/or exposure. The Getis-Ord G-statistic was used to assess spatial heterogeneity of combined infections and serological markers.A national cross-sectional household survey was undertaken from December 2008 to January 2009. A finger prick blood sample was taken from randomly selected participants of all ages to examine for parasitaemia using rapid diagnostic tests (RDTs) and confirmed using Polymerase Chain Reaction (PCR). Blood spots were also collected on filter paper and subsequently used to evaluate the presence of serological markers  for all ages; 6.9% (46/649) in children under the age of five years; and 14.2% (76/510) in the oldest age group (≥ 50 years). The combined infection and/or antibody prevalence was 10.5% (550/4769) and varied from 8.1% to 14.1% but overall regional differences were not statistically significant . Increasing age (p < 0.001) and decreasing household wealth status (p < 0.001) were significantly associated with increasing combined P. falciparum infection and/or antibody prevalence. Significant P. falciparum hot spots were observed in Dikhil region.A total of 7151 individuals were tested using RDTs of which only 42 (0.5%) were positive for P. vivax and human population movement across the sub-region remain to be properly established.Malaria transmission in the Republic of Djibouti is very low across all regions with evidence of micro-epidemiological heterogeneity and limited recent transmission. It would seem that the Republic of Djibouti has a biologically feasible set of pre-conditions for elimination, however, the operational feasibility and the potential risks to elimination posed by  In Africa, some countries in the North have achieved malaria elimination in the last 30 years  and the majority of the region is largely regarded as malaria free despite a few disputed residual foci in Algeria and Egypt. Elimination, however, poses a major challenge in the majority of countries in Africa owing to the intrinsically high transmission intensity within each country's national borders ,5.A recent systematic analysis of available national data has defined possible biological threats  and operational threats  to elimination across 99 malaria endemic countries . Within National Malaria Indicator Surveys (MIS) have been established largely to provide milestone measurements for reaching endemic control ambitions in high to moderate transmission countries, with parasite prevalence as the main transmission indicator. In low transmission areas this indicator is not efficient ,11. Here2 located at the southern reaches of the Red Sea on the Horn of Africa, bordered by Somaliland region of Somalia in the South, Ethiopia in the West and South and Eritrea in the North. It was the last French territory on mainland Africa to gain independence in 1977 with important strategic economic and political value as a port in the Gulf of Aden. The significance of the port at Djibouti for the Ethiopian economy was recognized at the end of the 19th Century when the Emperor declared Djibouti an official outlet of Ethiopian commerce that prompted the development of the Addis Abba-Dire Dawa-Djibouti Railway  or the Afars . Other smaller groups exist including people of Arab origin and refugees who migrated into the country during the 1980's and 1990's from the war-ravaged neighboring countries. The population is spatially over-dispersed with 60% of the entire country residing in Djibouti Ville and its suburbs. Five secondary urban settlements exist outside the main city at Arta, Obock, Tadjourah, Ali Sabieh and Dikhil  to >= 90% or 60% of children below the age of 5 years protected by an ITN by 2010; introduction of larvivorous fish and Abate for larval control; and urban fogging with insecticides; and case management with first line recommended therapy for uncomplicated malaria as artesunate and sulphadoxine-pyrimethamine combination (AS+SP) combined with increased diagnostic capacities in all health facilities. In addition monitoring systems of health and inter-country and cross-border collaboration were to be established . Therefore confirmation of RDT positive and 50 randomly selected negative cases was undertaken using Polymerase Chain Reaction (PCR) protocols for 2 spot from the filter paper was excised using a fresh scalpel blade per sample and put into a 1.5 ml pre-labeled tube at room temperature. The QIAamp® DNA Mini Kit  was used to extract DNA according to the manufacturer's instructions. For the RDT positive samples a second extraction was performed using the ABI6500 semi-automated extraction system. Five positive control samples were produced by spotting a parasite blood culture at 40% haematocrit and 1% parasitaemia. Five negative controls were made from non-infected blood in a similar approach. Real-time PCR using a P. falciparum specific probe for the the small subunit of rRNA (18s) gene was carried out using the forward and reverse primers 5'-TGCCGACTAGGTGTTGGATG-3' and 5' GCCCCAGAACCCAAAGACTT 3' respectively, and a TaqMan Minor Groove Binder (MGB) probe 5'-FAM-CATCTTTCGAGGTGACTT-MGB-TAMRA 3' labeled with 5'FAM (6-carboxyfluorescein) and 3'TAMRA (6-carboxytetramethyl-rhodamine) as the reporter and quencher, respectively. 2 μl of DNA was added to each reaction in a final volume of 20 ml with 1000 nM each of the forward and reverse primers, 250 nM of the TaqMan probe, and 10 μl of TaqMan Universal master mix . The PCR cycling steps were as follows: 50°C for 2 minutes, 95°C for 10 minutes, and 45 cycles of 95°C for 15 seconds and 60°C for 1 minute. All positive controls had critical threshold (tC) values below 34 cycles. A cutoff of 38 cycles was used to define positive samples.A 1-3 mmsvy command. Visual display of all cluster level attributes was undertaken using the GPS coordinates for each cluster and displayed using ArcGIS 9.3 (ESRI Inc. USA). Inland water bodies were assembled from the Global Lakes and Wetlands Database (GLWD) developed by the World Wildlife Fund . The combined PCR validated P. falciparum RDT positivity and sero-prevalence showed some evidence of between region variations with Djibouti Ville showing the lowest rates (8.1%). These regional differences were overall not statistically significant. This study therefore shows that the risks of P. falciparum malaria are low across the Republic of Djibouti but majority of clusters surveyed, 102/129 (79%), reported at least one individual who was positive for P. falciparum antibodies and/or peripheral blood infection . Our findings support the biological possibility for elimination reported elsewhere . ITN covP. falciparum infection or sero-prevalence in this study, human population movement is likely to be a significant challenge to elimination in the Republic of Djibouti. Several authors have highlighted the role played by the rail route to Ethiopia in the recent re-emergence of malaria transmission in Djibouti . This and the proposed future studies will substantially improve our understanding of malaria transmission in Djibouti, but without a carefully defined plan, staff or funding elimination of malaria in Djibouti remains only a biological possibility. The threshold of parasite prevalence seen as the benchmark for deciding whether to move on to malaria elimination or sustain low endemic control is seen as 1% parasite prevalence [The results of the survey support the apparent low case reporting of e 1970's ,16. P. fmination . This womination . At presevalence ,11. But evalence . An alteevalence ,26. To rThe authors declare that they have no competing interests.AMN was responsible for study design, data cleaning, analysis, interpretation, drafting and production of the final manuscript. MBM was responsible for the serological analysis of samples in the laboratory and data assembly and contributed to the final manuscript. CKM was responsible for the analysis of the optical density data, interpretation of results and contributed for the final manuscript. MAO, HHG, IAA contributed to the survey design, data assembly and cleaning and contributed to final manuscript. MN was responsible for the polymerase chain reaction analysis of all positive and selected negative samples and contributed to the final manuscript. JC and CJD were responsible for the development of ELISA methods, interpretation of results and contributed to final manuscript. MJM contributed to the statistical analysis of the optical densities, was responsible for the PCR of positive and negative samples and contributed to the final manuscript. RWS was responsible for overall scientific management, analysis, interpretation and preparation of the final manuscript. All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/11/121/prepub


Bupropion, which is widely used in patients with depressive disorder, may cause allergic reactions. However, the real prevalence of these side effects may be overlooked and underreported due to the delayed onset phenomenon.This study aimed to estimate the real incidence of bupropion-induced urticaria and clarify the delayed onset phenomenon.We conducted a nationwide cohort study between 2000 and 2009 using Taiwan’s National Health Insurance Dataset. Among 65,988 patients with depressive disorders, we identified new users of bupropion with depressive disorders  and matched them at a ratio of 1:4 regarding age and sex . The risk of urticaria was compared between the two cohorts.p = 0.001). The occurrence of urticaria in the bupropion cohort were more frequent on Days 15–28 than Day 1–14 (p = 0.002). Cox proportional hazards model showed that a history of urticaria was an independent risk factor for developing bupropion-induced urticaria.The risk of urticaria occurrence was higher in bupropion users than in matched controls within 4 weeks of starting the medication (risk ratio 1.81; 95% confidence interval 1.28–2.54; Of the antidepressants, bupropion may pose a higher risk of drug-induced urticaria, and this condition might be ignored due to the delayed onset phenomenon. Depressive patients with a history of urticaria are at higher risk of the adverse drug reaction. This study emphasizes the need for increased clinical awareness of this adverse outcome to bupropion use. Bupropion hydrochloride is a popular psychotropic drug that inhibits norepinephrine and dopamine reuptake with minimal effect on serotonin. It is prescribed to millions of patients worldwide for the treatment of major depressive disorders and bipolar depression. In 1997, bupropion was approved by the U.S. Food and Drug Administration (FDA) for use as a smoking cessation aid.The U.S. prescribing information for bupropion discloses serious allergic reactions as potential side effects, including urticaria, angioedema, erythema multiforme, Stevens-Johnson syndrome, and even anaphylactic shock. However, these allergic reactions have been rarely reported with bupropion. Two larTo date, only case reports have described bupropion-induced allergic reactions, and it is important to note that clinical features of delayed onset urticaria seems to be present in most of these patients-10. SomeTo address these inadequacies in the literature and to assess the pattern of bupropion-induced urticaria, we designed a nationwide population-based study to investigate the actual incidence and timing of new onset urticaria in patients with depression who receive bupropion prescriptions.  The Taiwan National Health Insurance (NHI) program offers a comprehensive, unified, and universal health insurance program to all citizens. The NHI program covers more than 96% of the country’s population and has contracted with 99% of all hospitals and clinics in Taiwan. The NHI We conducted a retrospective cohort study from January 1, 2000 to December 31, 2009. The presence of depressive disorders was defined as any defined in the International Classification of Diseases, 9th revision, and the clinical modification (ICD-9-CM) codes 296.2X-296.3X, 300.4, and 311.X,14. We aFor the study cohort, we identiﬁed 65,988 patients with depressive disorders who had received a first prescription for antidepressants (ATC code N06A) available in Taiwan between January 1, 2000 and December 31, 2009. Patients with a new prescription of bupropion were assigned to the bupropion cohort. Patients who had received bupropion before 2000 or under 20 years of age were excluded. We used the date on which the bupropion treatment was ﬁrst prescribed as the index date. Within the same observational period, for each of the 2,839 depressive patients taking bupropion, 4 insured people in the comparison cohort with other antidepressants use matched for age, sex, comorbidities, and index date were selected. The same exclusion criteria were applied to the matched comparison cohort. The other antidepressants used in the comparison cohort included fluoxetine, paroxetine, sertraline, citalopram, escitalopram, fluvoxamine maleate, venlafaxine, duloxetine, milnacipran, mirtazapine, trazodone, moclobemide, imipramine, amitriptyline, doxepin, maprotiline, clomipramine, dothiepin. The comparison group included 11,356 patients. In our study, the follow-up began on the index date and will end on 4 weeks later. We defined patients with an urticaria occurrence as those under ICD-9-CM codes 708.x within 4 weeks of receiving a bupropion prescription. Patients with recent urticaria within one month before receiving the new prescription of bupropion were excluded from the study. A history of urticaria was defined as urticaria occurring at least one month before the index date. The medical history could be traced because the NHIRD in Taiwan was established in 1996.Patient comorbidities at baseline were identiﬁed. Comorbidities included autoimmune diseases (ICD-9-CM code 279.x), liver diseases (ICD9-CM codes 570-573), diabetes mellitus (ICD-9-CM codes 250), chronic kidney diseases (ICD-9-CM codes 580-593), and HIV/AIDS (ICD-9-CM code 042).Each study patient was tracked until one of the following conditions was met: a diagnosis of urticaria (ICD-9-CM codes 708.x), follow-up was censored at the time of loss to follow-up, death, the patient withdrew from the NHI, or the follow-up period elapsed (4 weeks after index date).In addition, to clarify whether other antidepressants could have the same delayed onset phenomenon on urticaria occurrence, we compared urticaria occurrence in patients receiving a new bupropion prescription with patients receiving new prescriptions for all other antidepressants available in Taiwan within the next four weeks as a further analysis. 2 test for sex and baseline comorbidities. The cumulative incidence of urticaria for the two cohorts were calculated by the Kaplan-Meier method, with the log-rank test being used to examine differences between cohorts. Cox proportional hazards model was used to identify risk factors for urticaria occurrence in depressive patients with bupropion prescription within four weeks. We implemented in both the univariate and multivariable fashion to identify risk factors for the urticaria occurrence after bupropion use. Variables included in the model were sex, age, comorbidities, and history of urticaria. The qualifying criterion for inclusion in the multivariate analysis was a result in the univariate-analysis with a P value of less than 0.1. In constructing Cox models, the follow-up began on the index date. The study patients who withdrew (including those who died) from the NHI program were censored. If the patient did not leave the NHI program and encountered no urticaria occurrence, the date of censoring was the date of the end of follow-up. We validated the Cox regression model by checking whether the assumption of proportionality held. The results of Scaled Schoenfeld Reiduals test shown the proportionality of hazards assumption was met in this analysis  extracted and computed data. Microsoft SQL Server 2005  was used for data linkage, processing, and control sampling. IBM SPSS  and SAS statistical software  were used for all statistical analyses. Results of comparisons with a  Characteristics of patients in the bupropion and comparison cohort are shown in p = 0.001) (p < 0.001), but not in patients over 40 years of age. The use of bupropion was associated with a higher risk of urticaria occurrence in both males and females. = 0.001) . We alsop < 0.001).  As shown in p < 0.001) was the only independent risk factor for urticaria occurrence following bupropion use. We next performed univariate and multivariable analyses to predict urticaria development in the bupropion cohort . We founp = 0.002). Among all antidepressants, the delayed-onset phenomenon was only observed in bupropion users. To clarify whether other antidepressants could have the same delayed onset phenomenon on urticaria occurrence, we compared the cumulative incidences of urticaria in depressive patients with patients receiving all other types of new antidepressant prescriptions in Taiwan . Bupropip = 0.001), but not in patients over 40 years of age. The higher risk of urticaria occurrence in the bupropion cohort was significant in males but not in females . In addition, the delayed onset trend of urticaria occurrence was still found in this subanalysis . Detailed results are shown in the In this subgroup of dermatologist-diagnosed urtiacria among both cohorts, the higher risk for overall urticaria occurrence was similar to the results of the urtiacria patients diagnosed by general practitioners in the bupropion cohort. The bupropion use was still associated with higher urticaria risk in patients under 40 years of age . Besides, the frequency of antibiotics and anticonvulsants co-prescriptions with antidepressants is relatively low during a short follow-up period (4 weeks) in our study. Third, a number of other potential confounding factors that might affect urticaria risk were not available in the reimbursement data used in this study, such as psychosocial stresses, foods, family history of urticaria, recent travel history, exposure to environmental stimulants and smoking. {StocklIn conclusion, our study has found that patients with depressive disorders who received bupropion were at an unusually higher risk for delayed onset urticaria. Based upon our data, we suggest that more attention should be focused on the bupropion-induced urticaria in patients with depressive disorders and urge caution over the delayed onset phenomenon, especially in patients under 40 years of age and with past history of urticaria, in order to avoid more severe drug allergies as a result of prescribing the same drugs. Although more research is needed, this article serves to broaden physicians’ knowledge base when prescribing bupropion. Table S1Scaled Schoenfeld residuals test of proportional hazards.(DOC)Click here for additional data file.Table S2Incidence of dermatologist-diagnosed urticaria occurrence in depressive patients within first 4 weeks.(DOC)Click here for additional data file.Table S3Comparisons of early and delayed onset dermatologist-diagnosed urticaria occurrence.(DOC)Click here for additional data file.


Plasmodium infection.The effects of helminth co-infection on malaria in humans remain uncertain. This study aimed to evaluate the nature of association of intestinal helminths with prevalence and clinical outcomes of Plasmodium and intestinal helminth infections were diagnosed using Giemsa-stained blood films and Kato-Katz technique, respectively. Haemoglobin level was determined using a haemocue machine.A cross-sectional study involving 1,065 malaria suspected febrile patients was conducted at Dore Bafeno Health Center, Southern Ethiopia, from December 2010 to February 2011. Plasmodium parasites . Among 702 patients who provided stool samples, 53.8%, 31.6% and 19.4% were infected with intestinal helminths, Plasmodium alone and with both Plasmodium and intestinal helminths, respectively. The prevalence of infections with Ascaris lumbricoides (A. lumbricoides), Trichuris trichiura (T. trichiura), Schistosoma mansoni (S. mansoni) and hookworm (9.8%) were 35.9%, 15.8%, 11.7% and 9.8%, respectively. Out of the 222 (31.6%) Plasmodium infected cases, 9 (4.1%) had severe malaria. P. falciparum infection was more common in febrile patients infected with A. lumbricoides alone (21.3%), T. trichiura alone (23.1%) and S. mansoni alone (23.1%) compared to those without intestinal helminth infections (9.3%) . Prevalence of non-severe malaria was significantly higher in individuals infected with intestinal helminths than in those who were not infected with intestinal helminths . The chance of developing non-severe P. falciparum malaria were 2.6, 2.8 and 3.3 times higher in individuals infected with A. lumbricoides alone, T. trichiura alone and S. mansoni alone, respectively, compared to intestinal helminth-free individuals . The odds ratio for being infected with non-severe P. falciparum increased with the number of intestinal helminth species (p<0.001). Mean Plasmodium density among intestinal helminth infected individuals was significantly increased with the number of intestinal helminths species (p=0.027). Individuals who were co-infected with different species of intestinal helminths and Plasmodium showed lower mean haemoglobin concentration than individuals who were infected only with Plasmodium.Among 1,065 malaria suspected febrile patients, 28.8% were positive for A. lumbricoides, T. trichiura and S. mansoni were positively associated with P. falciparum infection. However, further studies are required to investigate how these helminths could contribute to increased prevalence of P. falciparum infection.Infections with  Plasmodium and helminth co-infections are prevalent in tropical countries, the effect of their interactions remains unclear [Plasmodium infection [P. falciparum malaria and intestinal helminths infection [Plasmodium density and intensity of hookworm infection [Although  unclear . Some stnfection -4, increnfection , decreasnfection  and incrnfection -9. Crossnfection ,11. Posinfection .On the other hand, evidence from other studies showed that helminth infections may protect from malaria or related clinical outcomes by suppressing acute clinical manifestations ,14, paraPlasmodia stages and species or altered immune response because of helminth co-infections [Plasmodium, epidemiological setting of the study area and other confounding factors could contribute for the contradictions.Still other studies reported the absence of significant association between helminth infection and malaria ,20. Thesfections . In addiP. falciparum was less common (only 4.0%), and infection with S. mansoni was absent [Plasmodium infected cases, and factors such as age, sex, nutrition and socioeconomic status were not considered in the analysis. Thus, the current study was conducted to assess the nature of association of intestinal helminths with prevalence and clinical outcomes of Plasmodium infection among malaria suspected febrile individuals attending Dore Bafeno Health Center, Southern Ethiopia.In Ethiopia, previous similar study was conducted in an area where infection with s absent . In addiA cross-sectional study involving 1,065 febrile patients (age range= 1 to 82 years) was conducted at Dore Bafeno Health Center, Sidama Zone, southern Ethiopia, December 2010 to February 2011. The health center is located in Dore Bafeno District, about 283 km south of Addis Ababa, at about 1,708 m above sea level and 7° 5' N and 38° 29 E. It has a mean annual rainfall of 80.4 mm (it varies from 15 mm to 130 mm) and mean annual temperature of 23.2°C (it varies from 21.5°C to 25°C). The study participants were malaria suspected febrile patients who did not take anti-malarial drug within the last two weeks. Almost all the study participants belonged to the Sidama ethnic group, residing in rural area with similar life style and earning their living as mixed-farming. Malaria is unstable in the area and occurs mainly from October to December following the heavy rainy season and from April to May following the light rain season .Clinical data including auxiliary body temperature, blood pressure, coma status, breathing status and prostration were collected by physicians. Clinical data together with laboratory results were used to group malaria cases as severe and non-severe . Socio-dApproximately, 20μl finger prick blood was collected from each febrile patient using a plastic capillary tube and 10 μl of the blood was used to prepare thick and thin blood films on a single slide for the diagnoses and count of malaria parasitaemia . The sliT. trichiura, S. mansoni and A. lumbricoides infections were quantified at the Aklilu Lemma Institute of Pathobiology (ALIPB), Addis Ababa University. Average egg counts of the two slides was multiplied by 24 to obtain egg counts per gram of stool for each of the study participants and classes of intensity was determined for different helminth species as per the WHO recommendation [Fresh stool specimens were collected from each study participant who also gave blood sample for malaria diagnosis. The stool specimens were prepared for microscopic examination using double Kato slides . Quantitendation .P. falciparum and P. vivax infection were treated with coarteum and chloroquine, respectively. Individuals who were found positive for the soil-transmitted helminths (STHs) were treated with 400mg of albendazole while praziquantel was used to treat those who were positive for S. mansoni, Taenia saginata (T. saginata), and Hymenolepis nana (H. nana). The study participants were treated free of charge. Since the study participants were selected from patients coming to the health center seeking medical treatment for malaria, they were not compensated for extra incentive to replace their transport cost or time lost from work was not given.This study was conducted after obtaining ethical clearance from the Institutional Review Board (IRB) of Aklilu Lemma Institute of Pathobiology, Addis Ababa University. As the majority of the population in the study area was illiterate the IRB endorsed oral consenting of the study participants. Permission to conduct the study was also obtained from Awassa Zuria District Health Office and Dore Bafeno Health Center. Detailed explanations about the aims, procedures, potential risks and benefit of the study were given to physicians of the health center. Individual oral consent was obtained in local language by the physicians of the health center during clinical examination and only voluntarily consented individuals were included in the study. For children younger than 18 years, assent was obtained from parents or guardians. Treatment was made based on the national protocol for treating malaria and helminth infections. Individuals who were found positive for 2-test and one-way ANOVA were used to compare malaria prevalence and mean parasitaemia or egg intensity values, respectively across categories of categorical variables. Univariable and multivariable logistic regression analyses were used to test for an association between intestinal helminths infection and prevalence of non-severe malaria or malaria related anaemia. Multiple linear regression analyses was used to evaluate differences in the means of Plasmodium density and haemoglobin level between Plasmodium-helminth co infected and those who were infected with Plasmodium alone. The analysis was adjusted for age, sex and nutritional status [Plasmodium infections, or related outcomes was evaluated. Since the study population was homogenous in their socio-economic status differences in terms of socioeconomic status were not considered during the analysis. The distribution of parasite density was skewed and square root transformation was used to make it symmetrical before using linear regression to evaluate the effect of pre-specified risk factors on this outcome. Results were considered significant whenever p-value was less than 5%.Data was computerized using Epi-data version 3.1 software  and analyzed using STATA version 11 . Pearson χl status ,30, whilPlasmodium parasites . P. falciparum infection alone or mixed infection with P. vivax was also higher in the 5–15 years old children compared to in individuals older than 15 years (p<0.01). Infection with P. vivax alone was higher (p<0.001) in the under five children and in the 5–15 years age group compared to those individuals older than 15 years. There was no significant difference in the percentage of P. falciparum and/or P. vivax infection between males and females.Among 1,065 malaria suspected febrile patients (Mean age=18.6 years), 306 (28.8%) were positive for A. lumbricoides (35.9%), T. trichiura (15.8%), S. mansoni (11.7%), hookworm (9.8%), T. saginata (3.9%), H. nana (1.4%) and Enterobius vermicularis (E. vermicularis) (0.9%). Of the 378 intestinal helminth infected individuals, 35.8%, 13.3%, 4.3% and 0.6% had single, double, triple and quadruple infections, respectively. Intestinal helminth infection was most prevalent among individuals of age 5–15 years (63.2%). A. lumbricoides and T. trichiura infections were higher among individuals in 5–15 years age group than among individuals of age >15 years.Among 1,065 febrile patients examined for malaria, 702 were volunteered to provide stool samples for the diagnosis of infection with intestinal helminths. There was no significant difference in baseline demographics between those who provided stool sample and those who did not (data not shown). Out of 702 patients examined, 53.8% were positive for at least one intestinal helminth species  for S. mansoni, 107 epg  for hookworm and 101 epg  for T. trichiura. Intensity of A. lumbricoides infection showed age related pattern with the highest  mean intensity being observed among individuals of age group 5–15 years (mean epg=3113.7) followed by individuals of age group <5 years (mean epg=2410.6). Mean intensity of hookworm infection was higher  among individuals of age <5 years (mean epg=648) followed by those individuals whose age ranges from 5–15 years (mean epg=69.6).Mean intensity of infections were 2345 egg per gram {epg}  for Plasmodium alone, and 136 (19.4%) with both Plasmodium and intestinal helminths  . Prevalence of P. falciparum infection was significantly higher in individuals infected with any intestinal helminth (s), A. lumbricoides alone, T. trichiura alone, S. mansoni alone compared to those who were not infected with intestinal helminths (9.3%)   were infected with intestinal helminths alone, 86(12.3%) with 4.5% werePlasmodium infected patients, 14 showed one or more sign and symptoms of severe malaria . Nine severe malaria cases were volunteered to provide stool samples for the diagnosis of helminth infection and 7 of them were co-infected with intestinal helminths like A. lumbricoides, T. trichiura and hook worm.Out of 306 P. falciparum mono-infection was higher among individuals infected with intestinal helminth than non-infected with intestinal helminth . The ORs changed in parallel with the number of intestinal helminth species infection (p<0.001). Individuals infected with A. lumbricoides alone (AOR=2.6), T. trichiura alone (AOR=2.8), S. mansoni alone (AOR=3.3), any intestinal helminth (AOR=2.9), T. trichiura and A. lumbricoides (AOR=3.1), and T. trichiura, A. lumbricoides, S. mansoni and hookworm (AOR=4.0) had increased odds of non-severe P. falciparum mono-infection compared to those who were not infected with any intestinal helminths  . Similarly, infection with hookworm alone was not significantly associated with any of non-severe P. falciparum and/or P. vivax infection.Of the 292 non-severe malaria cases, 213 patients were examined for intestinal helminth infection and 61.0% were positive. The odds of non-severe malaria infection was higher in individuals infected with intestinal helminth compared to intestinal helminth-free individuals {adjusted Odds Ratio (AOR) =1.57, 95% CI= 1.12, 2.21}. Similarly, the odds of non-severe Plasmodium density was significantly higher in individuals co-infected with four different intestinal helminth species  compared to those who were free from intestinal helminth infection . Similarly, there was an increasing trend in mean Plasmodium parasitaemia among individuals who were infected with T. trichiura alone, S. mansoni alone, A. lumbricoides alone and any two or three of these species compared to individuals who were not infected with any intestinal helminth species. The mean Plasmodium density among intestinal helminth infected individuals increased significantly as the number of intestinal helminth species increased . However, patients infected with hookworm alone showed slightly lower mean Plasmodium parasitaemia compared to individuals who had no intestinal helminth infection.Data regarding the effect of intestinal helminths co infection on malaria-related outcomes are summarized in Table A. lumbricoides, hookworm, S. mansoni, T. trichiura alone or in combination and Plasmodium showed lower mean haemoglobin levels compared to malaria cases without intestinal helminths infections. However, the mean differences were not significant in the multivariable model. Prevalence of anaemia was higher among individuals co-infected with both intestinal helminths and Plasmodium compared to those with Plasmodium infection alone. Individuals co-infected with Plasmodium and four different intestinal helminths were almost 9 times more likely to be anaemic compared to those with Plasmodium infection alone .Individuals co-infected with Plasmodium density and intensity of intestinal helminth infections . In S. mansoni and T. trichiura infected patients, the mean parasitaemia increased as the class of intensity of the helminths increased; nonetheless the regression coefficients remain non-significant after adjusting for the effects of age, sex, nutrition, and concurrent infection by other species of helminth. Conversely, the mean parasitaemia of individuals with moderate intensity of A. lumbricoides infection was relatively low compared to individuals co infected with light intensity of A. lumbricoides and Plasmodium. For hookworm infection, all the infected cases were of light intensity class (data not shown).There was not significant correlation between the mean Plasmodium was negatively correlated with intensity of intestinal helminths. However, the coefficients for the association were low. Similarly, although the mean differences were not significant, haemoglobin levels decreased more in Plasmodium infected individuals co-infected with heavy intense S. mansoni or moderate intense T. trichiura infections compared to individuals with light intensity of infections with the respective intestinal helminths (data not shown).The reduction in mean haemoglobin level among individuals co-infected with intestinal helminths and P. falciparum malaria was significantly higher in individuals infected with intestinal helminths, particularly in those with A. lumbricoides alone, T. trichiura alone or S. mansoni alone compared to individuals without intestinal helminths. Similarly, prevalence of non-severe malaria  was significantly higher in individuals infected with intestinal helminth or A. lumbricoides alone compared to individuals without intestinal helminths. These findings are similar with previous finding in children [P. falciparum malaria was also previously reported in helminth infected individuals [In this study, the likelihood of being infected with non-severe children ,11 and achildren . Increasividuals ,7,8.Plasmodium and intestinal helminth co-infected patients compared to those with only Plasmodium infection. There was a non-significant decreasing trend in mean haemoglobin levels as the number of intestinal helminths species per an individual increased. Similarly, the association of anaemia was slightly higher in individuals co-infected with Plasmodium and intestinal helminth compared to malaria cases without having intestinal helminth infections. Intestinal helminths and malaria reduce haemoglobin level differently [In concordance with previous reports ,31, a referently ,33, implPlasmodium density was slightly higher among intestinal helminth infected individuals compared with intestinal helminth free individuals. These results together with the high odds of anaemia (or decreased mean haemoglobin levels) and non-severe P. falciparum and/or P. vivax infection give the impression of a positive association between intestinal helminth infection and non-severe malaria. This association between prevalence or outcomes of malaria and intestinal helminth infections seemed to increase with an increase in the number of intestinal helminths species. This is in agreement with previous reports from Ethiopia [Plasmodium density and P. falciparum infection, respectively increased with the number of intestinal helminth species.Mean Ethiopia  and ThaiEthiopia  where thPlasmodium[Plasmodium during concomitant infection [The present findings appear to substantiate the hypothesis that clinical malaria can be aggravated during concomitant infection of intestinal helminths and lasmodium. Worms alasmodium. Helmintlasmodium. Additionfection . Helmintnfection  promotinPlasmodium density was less associated with intensity of helminth infections and results regarding mean differences of Plasmodium density between intestinal helminth free and those infected with different intestinal helminth species were contradictory. Compared to intestinal helminth free malaria cases, the mean Plasmodium density of individuals infected with A. lumbricoides alone, S. mansoni alone or T. trichiura alone was slightly higher, but lower in the case of single hookworm infection. These controversial results on mean Plasmodium changes could have arisen due to the dominancy of light intense helminth infection among cases or complex nature of helminth and Plasmodium interaction when co-exist in a host.However, P. falciparum and/or P. vivax infection. This is in agreement with previous reports from Uganda [P. vivax mono-infection and infection with different intestinal helminth species which agrees with the observation in Thailand [Plasmodium species [Individuals with hookworm infection alone were neither protected nor associated with increased non-severe m Uganda  and Zairm Uganda , but in m Uganda . The lowThailand . This co species .Previous studies documented negative ,17-19 anPlasmodium species. In addition, the effect of confounding factors such as age, sex, nutrition status, socioeconomic factors and other infections  were not usually considered in previous studies. It is well known that, level of endemicity , local climate, traditional and modern health care practices as well as community health seeking behaviors vary from country to country and even from district to district. These variations could also bring substantial differences in helminth-malaria interactions and hence a result from one country/district might not be necessarily generalized or interpreted in another country/district where the above factors are significantly different.Variation in inferences about the nature of malaria and helminth interaction among reports could be due to differences in the nature of the study participants , study design, methodology and the way data was analyzed and interpreted. In most previous studies, data analysis did not separate the effect of different helminth or Participants of the current study were more homogenous in that they were largely (>90% of the cases) similar in their life style, education level (illiterate), income level (average) and means of making a living (agriculture), place of residence  and ethnicity (Sidama). However, slight differences in socioeconomic status might have existed among the cases which could partly affect the current data. In addition, cross-sectional nature of the study design has been a limitation to make conclusion about the risk or incidence of malaria due to previous status of helminth infection.A. lumbricoides, T. trichiura and S. mansoni were positively associated with P. falciparum infection. However, further studies are required to investigate how these helminths could contribute to increased prevalence of P. falciparum infection.In the current study, infections with The authors declare they have no competing interests.AD: conceived the project idea and designed the study, collected, analyzed and interpreted the data. GM: involved in data analysis. AD, GM, ML, AA and BE involved in the drafting of the manuscript for important intellectual content. All authors read the draft manuscript and approved the final copy for submission.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/12/291/prepub


Plasmodium falciparum malaria. Few studies exist on immunohistochemical characterization of the placental inflammatory infiltrate. The current study was conducted in Gadarif hospital in an area characterized by unstable malaria transmission in eastern Sudan.Maternal immunity is thought to play a major role in the increased susceptibility of pregnant women to Ninety three placentae were investigated for malaria histological changes and immunohistochemical study for monocytes and macrophages (CD68).P = 0.047] especially in placentae with past malaria infections. Placental malaria infections and monocytes and macrophages cells infiltration were not different between primiparae and multiparae. There was no significant difference in the birth weight between the women with placental malaria infections/monocytes and macrophages cells infiltration and those who had no placental malaria infections/cellular infiltrations.While 1(1.1%), 2(2.2%) and 20(21.5%) of the 93 placentae had acute, chronic and past malaria infections, 70(75.2%) had no malaria infections. Monocytes and macrophage (CD 68) were detected in 29 (31.2%) of these 93 placentae. Significantly higher rate of monocytes and macrophage were detected in placentae with malaria infections [11/23 (47.8%) vs. 18/70 (25.7%); Significantly higher rate of monocytes and macrophage were detected in placentae with malaria infections. Neither placental malaria infections nor cellular infiltrates were associated with parity or lead to reduction of birth weight. Malaria during pregnancy is a major public health problem in tropical and subtropical regions; each year 25 million African women become pregnant in malaria endemic areas . PregnanPlasmodium falciparum-infected erythrocytes to syncytiotrophoblast leads to parasite sequestration in the intervillous space. The parasite adheres specifically to chondroitin sulfate-A expressed on syncytiotrophoblast [P. falciparum-infected placentae are characterized by an increase in inflammatory cells in the intervillous space [During pregnancy, adhesion of phoblast . The incphoblast ,9. Howevphoblast , the immphoblast . The infphoblast ,13. Histus space . The plaus space -17. Few us space . In the us space .P. falciparum parasites per 200 white blood cells was counted and double-checked blindly by an expert microscopist. Maternal haemoglobin concentrations were estimated by HemoCue haemoglobinometer .A cross sectional study was conducted in Gadarif Maternity Hospital during October 2009. Ninety-three consecutive women with singleton pregnancy were approached to participate in the study. After signing an informed consent, obstetrical and medical history (age and parity) were gathered using questionnaires. The babies were weighed immediately following the delivery using electronic digital scale to the nearest 50g. Maternal, placental and cord blood films were prepared, the slides were Giemsa-stained and the number of asexual 3 sample was removed from the maternal surface in an off-center position, half the distance between the umbilical cord and the edge of the placenta. Once collected, each biopsy sample was placed in 25 mL of 10% neutral buffered formalin. All biopsy samples were kept at room temperature and were stored in Gadarif until transportation to Khartoum, where the histologic studies were performed. The placental biopsy samples were then processed and were embedded in paraffin wax, by standard techniques. In every case, paraffin sections 4 mm thick were stained with hematoxylin-eosin and Giemsa stain. Because the samples were fixed in buffered formalin, formalin pigment formation, which has similar optical characteristics and polarized light activity to malaria pigment was not detected [et al [The details of this have been shown before ,20. In sdetected : Placentd [et al : uninfechttp://www.ihcworld.com/products/IHC-Tek-Reagent.htm. In summary, 4-mm sections were deparaffinized and were hydrated through xylene and graded alcohols, and peroxidase was blocked for 5 min in 0.03% H2O2 containing sodium azide. Then the slides were incubated with the primary antibody for 40 min against CD68. The peroxidase-labeled polymer was then applied for 40 min. After washing in TBS, the slides were incubated with the diaminobenzidine substrate chromogen solution, washed in distilled water, counterstained with hematoxylin, washed, dehydrated, and mounted. A pressure cooker was used for heat-induced epitope retrieval with antibodies [Immunohistochemical analysis was performed in neutral formalin-fixed, paraffin-embedded tissue, using the IHC-Tek™ Avidin/Biotin Blocking Solution following the manufacturer's instructions tibodies . Monocyt2 were used to compare means and proportions between the groups, respectively.Data were entered in computer using SPSS for windows version 16.0 for analysis. Student's t-test and XThe study received ethical clearance from the Research Board at the Faculty of Medicine, University of Khartoum.The age of these 93 women ranged from 13-44 with the mean (SD) of 25.9 (7.3) years. Of these 93 women, 34 (36.5%) were primiparae. Only 12 (13.0%) gave history of using bed nets and none of these 93 women used malaria prophylactic treatment during the index pregnancy. There was one blood film positive in maternal and placental set. Of these 93 women, 47 (50.5%) were anaemic (haemoglobin < 11 g/dl) and 7 (7.5%) had low birth weight deliveries.P = 0.838. The rate of anaemia was not different between women with placental infections and those women who had no placental infections, 12/23 (52.2%) vs. 35/70 (47.3), P = 0.944. There was no significant difference in mean (SD) birth weight between the women with placental malaria infections and those who had no placental malaria infections, 2962.5(455.5) vs. 3035.7 (538.4) gm; P = 0.576.Placental histology showed that 1 (1.1%), 2 (2.2%) and 20 (21.5%) had acute, chronic and past malaria infections, respectively and 70 (75.2%) had no malaria infections. Of 34 primiparae, 8 (23.5%) vs. 15 (25.4%) of the 59 multiparae had placental malaria infection, 2. Of 34 primiparae, 11(32.3%) vs. 18 (30.5%) of the 59 multiparae, P = 0.853 had placental monocytes and macrophage cell infiltrations.The monocytes and macrophage cell infiltrations were detected in 29 (31.2%) of the placentae. The number of monocytes and macrophages ranged from one to fifteen cell with the geometric mean (SD) of 3.1(3.3) cells/mmP = 0.047, table P = 990) and the rate of low birth weight deliveries was not different between those who had placentae with monocytes and macrophages infiltrates and those who had placentae without cellular infiltrate, table Significantly higher rate of monocytes and macrophages infiltrates were detected in placentae with malaria infections, 11/23 (47.8%) vs. 18/70 (25.7%); This is the first study to investigate malaria placental histology and monocytes and macrophage cellular infiltrations in an area with unstable malaria transmission in Africa. The main findings of the current study were; most 20/23, 87.0%) of the placental infections were past infections which affect pregnant women regardless to their parity and had no effects on birth weight. Monocytes and macrophages cellular infiltrations were detected in 32.2% of the placentae. They were more predominant among placentae with past malaria infections irrespective to parity, associated with maternal anaemia and had no effect on birth weight. We have previously shown that placental malaria infections, hormonal and cytokines levels were not different between the primigravidae and multigravidae among pregnant women in eastern and central Sudan , 87.0% o. This obP. falciparum clearance and phagocytosis of the infected red blood cells. On the other hand these inflammatory cells might lead to functional damage in placental villi, and disturb feto-maternal exchange, leading to low birth weight [In neighboring Tanzania, it has been shown that, malaria parasitized placentae, especially in primigravidae, had the most significant increase in all inflammatory cellular types -except NK cells-with monocytes and macrophages representing the major population of the infiltrate . It has h weight ,13. The h weight ,20. The h weight . Due to Significantly higher rate of monocytes and macrophage were detected in placentae with malaria infections. Neither placental malaria infections nor cellular infiltrates were associated with parity or lead to reduction of birth weight.The authors declare that they have no competing interests.MMS and IA designed the study, GKA and MIE conducted the clinical work. MMS, AHM, MIE and AAM conducted the lab work. IA and GKA participated in the statistical analyses. All the authors approved the draft and the final paper.


The effects of iron interventions and host iron status on infection risk have been a recurrent clinical concern, although there has been little research on this interaction in pregnant women.Plasmodium falciparum parasitaemia and odds ratios of malaria risk.Cross-sectional and longitudinal analyses were undertaken to determine the association of whole blood zinc erythrocyte protoporphyrin (ZPP) with malaria parasitaemia in pregnant women attending antenatal and delivery care at Montfort and Chikwawa Hospitals, Shire Valley, Malawi. Prevalence of antenatal, delivery and placental malaria was assessed in relation to maternal ZPP levels. The main outcome measures were prevalence of peripheral and placental A total of 4,103 women were evaluated at first antenatal visit, of whom at delivery 1327 were screened for peripheral and 1285 for placental parasitaemia. Risk of malaria at delivery  was higher in primigravidae (p < 0.001), and lower  with use of intermittent preventive anti-malarials during pregnancy (p < 0.001). HIV infection was associated with increased malaria parasitaemia . Parasitaemia prevalence was lower in women with normal ZPP levels compared to those with raised concentrations at both first antenatal visit . Between first antenatal visit and delivery women who transitioned from raised  to normal ZPP values (at delivery) had lower peripheral parasitaemia prevalence at delivery compared to those who maintained normal ZPP values at both these visits . In regression analysis this difference was lost with inclusion of HIV infection in the model.P. falciparum parasitaemia and were probably secondary to malaria inflammation, rather than indicating an increased malaria risk with iron deficiency. It was not possible from ZPP measurements alone to determine whether iron deficiency or repletion alters malaria susceptibility in pregnancy.Raised ZPP concentrations in pregnancy were positively associated with  The effects of iron interventions and host iron status on infection risk have been a recurrent clinical concern  and thisTwo previous cross-sectional studies in Malawi  and TanzThe data for the analysis was collected between 1992–1995 in a large study, which described the pattern of malaria and anaemia during pregnancy and associations with pregnancy outcomes -15. The Cross-sectional and longitudinal analyses of whole blood zinc erythrocyte protoporphyrin (ZPP) were conducted in order to determine its association with malaria parasitaemia prevalence in pregnant women attending for antenatal and delivery care.Study enrolment was between March 1993 and July 1994 in Chikwawa District, Southern Malawi, a rural area with holo-endemic malaria transmission at the time of the study, and was located in two hospitals: Chikwawa District Hospital (CDH), a government hospital with free services, and Montfort Hospital (MH) 30 km away, which is a fee-paying mission hospital.All women attending the antenatal facilities of CDH or MH were screened at their first antenatal visit. Verbal informed consent was obtained. Women were encouraged to deliver in the hospital facilities and those who attended for delivery were screened a second time. Most women received two intermittent treatment doses of sulphadoxine-pyrimethamine  (IPTp-SP), one at the time of their first antenatal visit (if after the first trimester), and a second dose at between 28–34 weeks gestation .Plasmodium falciparum parasites against 200 white blood cells. A malaria smear was prepared from blood obtained from 1 cm placental villous incision.At enrolment a questionnaire was completed on obstetric history. Mid upper-arm circumference (MUAC) was measured in women at the mid-point of the left arm (mm) and height (cms) using a height board. Each subject was given a month’s supply of haematinics  at every antenatal visit and asked to visit monthly. At-delivery frequency of use of sulphadoxine-pyrimethamine was available from the antenatal card. A venipuncture blood sample was collected in an EDTA tube at first antenatal visit and also immediately prior to delivery. Within six hours of collection, haemoglobin (Hb) concentration was measured photometrically by conversion to cyanomethemoglobin (Biotron hemoglobinometer) using a standard control. Within 24 hours packed cell volume (PCV) was measured by micro-haematocrit), and ZPP in μg ZP/gHb (AVIV hematofluorometer). ZPP measurements were standardised against manufacturer control samples. Mean corpuscular haemoglobin concentration (MCHC) was calculated from Hb and PCV. Sera of mothers, whose babies were included in a separate infant follow-up study, were HIV tested after maternal informed consent and counselling. Maternal HIV infection was defined by presence of HIV antibodies determined by ICE* HIV-1.02  and confirmed by VIDAS HIV-2 test . At the time of the study, antiretroviral therapy was not available, nor Co-trimoxazole prophylaxis, so women did not receive these drugs. Malaria slides were prepared and stained with Giemsa and read counting asexual P. falciparum parasites on a blood smear from a venipuncture sample collected at first antenatal visit or delivery. Placental parasitaemia was a positive P. falciparum blood smear obtained from a sample collected following placental villous incision.Anaemia was defined as Hb <11 g/dl, severe anaemia as Hb <8 g/dl, and iron-deficient erythropoiesis as ZPP concentration > 2.7 μg ZP/g Hb ,16. Low Cross-sectional analyses measured malaria parasitaemia prevalence and ZPP concentration at either first antenatal visit or at delivery. Malaria prevalence was estimated for women with normal or raised ZPP levels. Longitudinal analyses classified ZPP transition categories , which occurred between the first antenatal visit and delivery, and related these to either placental or peripheral parasitaemia prevalence at delivery. Data were analysed using SPSS for Windows version 18. Discrete variables were compared using chi-square tests. For the longitudinal data set multivariate logistic regression was used to calculate odds ratios for factors associated with delivery malaria as the dependent variable . Factors included in the regression models were intermittent use of sulphadoxine-pyrimethamine in pregnancy (IPTp), gravidity, maternal HIV status, and longitudinal ZPP transition category.Ethical approval for the study was granted by the Malawi Health Science and Research Committee, and the Liverpool School of Tropical Medicine.The sample sizes available for the analysis are shown in Figure Prevalence of demographic and nutritional characteristics at first antenatal visit is shown in Table Table There were no differences in malaria prevalence at either first antenatal visit or delivery  between women with normal or low MCHC values, except for multigravidae at first antenatal visit with low MCHC, who had lower malaria prevalence, compared to those with normal MCHC .Prevalence estimates for peripheral or placental malaria at delivery for the four iron transition categories based on ZPP measurements are shown in Table Table For the longitudinal data set logistic regression analysis using two models was performed with malaria at delivery as the dependent variable. Models were based on the ZPP cut-off used to define iron deficiency. Factors included were gravidae, IPTp uptake ≥ one dose, and ZPP transition categories 1–4. The second model included HIV status as only a subsample of women (16 %) had known HIV status. Risk of malaria at delivery  was higher in primigravidae (p < 0.001), and lower  with IPTp use (p < 0.001). HIV infection was associated with increased malaria risk . There was lower risk of peripheral parasitaemia at delivery for women who transitioned from raised ZPP at first antenatal visit to normal ZPP values at delivery (p = 0.048) (ZPP category 2). This reduced risk remained in model 2, which included HIV status, but was no longer significant.This is the first analysis of ZPP as a potential iron biomarker of malaria risk in pregnancy. Use of ZPP transition categories allowed malaria risk to be assessed concurrently with longitudinal changes in this iron biomarker. Gestational iron status is influenced by antenatal haematinic use, iron bioavailability  and enteZPP reflects bone marrow iron requirements , and as In the cross-sectional analysis at first antenatal visit, malaria prevalence was higher in women with raised ZPP values for all gravidae classes except secundigravidae. At delivery, malaria prevalence  was also higher in woman with raised ZPP concentrations. This association in the cross-sectional study of higher malaria prevalence in women with elevated ZPP levels is the statistically most significant finding as in the longitudinal sub-group analyses sample sizes are much smaller. The association may result from inflammation increasing ZPP concentration, rather than indicating an association of increased malaria risk with maternal iron deficiency. This is because, based on MCHC, multigravidae at first antenatal visit who had low MCHC values also had lower malaria prevalence compared to those with normal MCHC, suggesting that iron deficiency may reduce malaria risk. This suggests ZPP is not a good biomarker for iron status in this population. A limitation of ZPP as an indicator of pregnancy iron status relates to factors known to elevate its concentration, including infection , impaireIn the present study gravidae who maintained normal ZPP concentrations at first antenatal visit and at delivery , had higher delivery malaria prevalence than those who transitioned from raised to normal ZPP values . This might suggest that continuous iron repletion during gestation was associated with higher delivery malaria risk, and not late gestational iron repletion. But women with continuous iron repletion did not have higher delivery malaria prevalence than women who transitioned to raised ZPP values at delivery , suggesting that if ZPP is accurately indicating maternal iron status then it is not discriminating malaria risk, as both chronically iron-replete and chronically iron-deficient women were at equivalent risk of malaria at delivery. However, if malaria-related inflammation raises ZPP concentration, independent of maternal iron status, then gestational transition to lower ZPP values would be associated with a reduction in malaria prevalence at delivery compared to women who show no change in ZPP concentration, as shown in this study.In regression analysis, peripheral or placental malaria was positively associated with primigravidae and HIV infection, and lower malaria risk was associated with uptake of one or more doses of IPTp-SP. These associations would be expected and their magnitude provides a useful gauge against which to assess malaria risk estimates related to ZPP categories. Transition to normal ZPP concentration was associated with reduced risk for malaria parasitaemia in peripheral or placental blood Table . These fA proportion of women in this population will have raised ZPP concentrations solely related to maternal iron deficiency and not related to inflammation. A limitation of this analysis is that without additional sensitive serum biomarkers of iron status it is not possible to discriminate this group from those with raised ZPP values due to inflammation alone. For this reason it is not possible to determine from ZPP measurements alone whether iron deficiency or repletion alters malaria susceptibility in these pregnant women. The proportions of women with raised ZPP values due to iron deficiency, or due to inflammation will also vary between gravidae as iron deficiency is generally more frequent in multigravidae .Analysis of ZPP as an iron biomarker from this large pregnancy data set provides evidence that elevation of ZPP concentration was associated with increased risk of malaria infection, and that this association was secondary to inflammation rather than indicating increased malaria risk with maternal iron deficiency. It was not possible from ZPP measurements alone to determine whether iron deficiency or repletion alters malaria susceptibility in pregnancy.ZPP, Zinc erythrocyte Protoporphyrin.The author(s) declare that they have no competing interests'.ES and GK analysed the data and assisted in writing the manuscript. BJB was principal investigator for the field study and contributed to data analysis and writing the paper. All authors read and approved the final manuscript.


Plasmodium falciparum-infected erythrocytes in brain microvasculature through cytoadherence to endothelium, is the hallmark of the definitive diagnosis of cerebral malaria and plays a critical role in malaria pathogenesis. The complex pathophysiology, which leads each patient to the final outcome of cerebral malaria, is multifaceted and thus, metrics to delineate specific patterns within cerebral malaria are needed to further parse patients.The sequestration of A method was developed for quantification utilizing counts of capillary contents  from histological preparations of brain tissue after death, and compared it to the standard approach, in which the percentage of parasitized vessels in cross-section is determined.vs CM2). A repeat CART analysis for the second cohort generated slightly different classification rules with more accurate subclassification, although misclassification still occurred.Within the initial cohort of 50 patients, two different observers agreed closely on the percentage of vessels parasitized, pigmented parasites and pigment globules (ICC = 0.795-0.970). Correlations between observers for correct diagnostic classification were high . When these methods were applied prospectively to a second set of 50 autopsy samples, they revealed a heterogeneous distribution of sequestered parasites in the brain with pigmented parasites and pigment globules present in the cerebellum > cortex > brainstem. There was no difference in the distribution of early stages of parasites or in the percentage of vessels parasitized across the same sites. The second cohort of cases was also used to test a previously published classification and regression tree (CART) analysis; the quantitative data alone were able to accurately classify and distinguish cerebral malaria from non-cerebral malaria. Classification errors occurred within a subclassification of cerebral malaria (CM1 The traditional measure of parasite sequestration in falciparum malaria, the percentage of vessels parasitized, is the most reliable and consistent for the general diagnosis of cerebral malaria. Methods that involve quantitative measures of different life cycle stages are useful for distinguishing patterns within the cerebral malaria population; these subclassifications may be important for studies of disease pathogenesis and ancillary treatment. Plasmodium falciparum, is responsible for nearly all of the severe morbidity and mortality in malaria-endemic areas [P. falciparum infections is the ability of infected erythrocytes, especially those containing parasites in the latter half of their life cycle, to adhere to the endothelium (i.e. cytoadherence) of capillaries and post-capillary venules [P. falciparum are rarely seen in the peripheral blood [Five species of malaria parasite can infect humans, but one, ic areas . A disti venules . This pral blood -6. EventP. falciparum infections range from asymptomatic parasitaemia to convulsions, coma and death. The mechanisms leading to central nervous system dysfunction in severe malaria have not been elucidated, but one hypothesis suggests that sequestration of parasitized erythrocytes in cerebral microvessels with resultant vascular congestion plays a crucial part in pathogenesis [The clinical syndromes associated with ogenesis ,8. To gaA clinicopathological study of fatal cerebral malaria is ongoing in Blantyre, Malawi. A reproducible and biologically relevant method of quantifying sequestration is necessary before comparisons between sites within an organ, or between patients, can be made. The traditional method is to establish, in a given tissue sample, the proportion of vessels which contain parasitized erythrocytes. This figure is then used as the basis for comparisons -13. ThisP. falciparum parasitaemia, and no improvement following effective treatment for seizures and hypoglycaemia. Comatose children who were aparasitaemic on four consecutive blood smears collected at six-hourly intervals were diagnosed as having non-malarial comas (NM Coma). Parasitaemic children without coma (Blantyre Coma Score >2 within two hours of admission) and a haematocrit <15% were classified as having severe malarial anaemia (SMA). Parasitaemic children without coma (Blantyre Coma Score >2 within two hours of admission) were classified as having other malarial illness (other). Parasitaemic children who died before a clinical diagnosis could be determined were grouped together as indeterminate. Parasitaemic children with an obvious non-malarial cause of coma were classified as non-malarial coma with incidental parasitaemia (NM Coma IP). All were treated initially with quinine; blood and antibiotics were administered according to standard criteria, and the treatment regimen was adjusted as the working diagnosis evolved [All patients were admitted to the Paediatric Research Ward  between February 1996 and June 2010. Demographic, historical, clinical, and laboratory information were recorded for the duration of admission. The Blantyre Coma Score  was usedIn the event of a death, a Malaŵian clinician met with key family members to discuss the possibility of performing an autopsy. When permission was granted, autopsies were performed in the mortuary of the Queen Elizabeth Central Hospital. The brain was cut directly at autopsy and blocks from standard regions were fixed in 10% pH neutral buffered formalin, processed per routine protocol, and embedded in paraffin. Sections cut at 5 μ were stained with haematoxylin and eosin.Histological sections of brain tissue were reviewed by pathologists  who were blinded to the clinical diagnosis and were classified as follows: sequestration only (CM1); sequestration and extravascular pathology including ring haemorrhages (CM2); and no evidence of sequestration/malarial pathology (CM3). The brain and remaining organs were evaluated systematically to determine cause of death.Fourteen cases with high rates of parasite sequestration (>50% of capillaries parasitized) in the cerebral microvasculature were included. Each was coded and an area was outlined on each slide where the grey matter was sectioned perpendicularly to the meningeal surface of the brain. The slide was secured on a standard microscope such that the outlined area of grey matter ran perpendicularly to the up/down or side/side direction of the microscope stage. Consecutive fields were counted, perpendicular to the meningeal surface, running toward the border between grey and white matter. At the completion of a run, the slide was moved across two field-widths followed by a second run back up to the meningeal surface. Microscope fields were assessed systematically until at least 100 cross-sectioned capillaries were identified and a run was complete : Parasites were counted as unpigmented if no pigment was seen within the erythrocyte and if the parasite generally occupied less than one third of the diameter of the erythrocyte : The common feature in these parasitized erythrocytes is the presence of malarial pigment which can vary in size from a small black dot to a less well defined larger mass of brown/black pigment : Malaria pigment appears as a single mass of ill-defined black pigment associated with a ‘ghost’ red blood cell, free in the vessel, or as multiple masses within intravascular white blood cells ; and fibrin: Identified on hematoxylin and eosin stain and quantified per 100 capillaries , and twice by three other individuals , following instruction by, and under the supervision of the experienced observer. All counts were made without knowledge of the results of other readings.Slides from the first 50 autopsies (MP1 to 50) of the series were prepared from the parietal lobe, midbrain, and cerebellum and the various parasite elements were counted as described above by a single observer (NF). For the parietal lobe, a second observer (DM) counted all parasite elements for all 50 cases. Modifications of the method were required in the occipital grey matter (data not utilized in this manuscript) where the runs were completed to the meningeal surface of white matter/grey matter junction when a minimum of 100 vessels had been assessed. In order to represent different layers within each region of the brainstem equally, areas on the para-sagittal plane of each slide were delineated, running in the longest possible axis. Runs were completed from the top to the bottom of the area to include a minimum of 100 vessels. In the cerebellum, an area of the slide was chosen in which the cerebellar folia were well represented, and consecutive fields were counted, running within the molecular layer of the folia until 100 vessels were examined. The results of this quantification (for NF only) were previously analysed using CART and published .For a subset of 28 of the first 50 cases, counts were performed on the parietal lobe, occipital lobe, midbrain, pons, medulla, and cerebellum by one observer (CP).Slides from the second 50 cases of the series (MP51 to 100) were prepared from one area of the cortex . The various parasite elements were counted as described by a single observer (DM). Counts were performed blinded to the clinical and pathological diagnoses.The complete counts for the first 50 cases  were compared between two observers (NF and DM) for each element by analysis of agreement (Bland-Altman plot), reproducibility , and correlation . Regression analysis for each variable was also performed. Additionally agreement for case classification as based on clinical and histopathological diagnosis of each observer was assessed via weighted Kappa co-efficients with corresponding P-values. Classification of the 50 cases was performed through %VP with cut-offs and rule defined by the previous CART analysis. For each observer, the cases were classified by the actual counts. Distribution of each element between the two observers was compared based on means and t-tests with p-values adjusted for multiple testing via Bonferroni approach (0.05/70).de novo CART analysis was performed using only the latter 50 cases and this method was run with five times cross-validation. CART software  was used for all tree analysis.All counted parasite elements were normalized to 100 vessels for all patients and the following variables were used as potential node differentiators:- UPP, PP, PG, TPG, fibrin, and %VP. For the purposes of clinical validity, all patients who met the clinical case definition of cerebral malaria were included as categories for classification, which included CM1, CM2, and CM3 (i.e. non-CM patients were excluded). Validation of the previous CART analysis was performed by classifying the latter 50 cases with the rules established by the first 50 cases. Briefly, the rule identified by the previous CART analysis classified as true CM (CM1 or CM2) patients who had greater than 23% of vessels parasitized, CM2 patients as those with greater than 55.5 pigment globules per 100 vessels, and as non-CM (CM3) patients as those less than 25% of vessels parasitized. A repeat Data from 100 patients were included; 71 patients met the clinical case definition of cerebral malaria of which 13 were pathologically classified as CM1, 39 as CM2, and 19 as CM3. The remaining 29 patients included four patients with SMA, 21 with non-malaria causes of coma or other non-malarial illnesses, and four indeterminate cases .A Bland-Altman plot of %VP was performed first and demonstrated excellent agreement between observers  were analysed across the pathological diagnoses, CM3 was significantly different  from CM1 and CM2 across all elements and CM2 had significantly more pigment  than CM1 . The levels of %VP were very low for subjects with CM3 and indistinguishable from control subjects. Using the previously established cut-off level of 23.3% for %VP, the sensitivity and specificity of %VP to discriminate subjects among patients meeting the clinical case definition  was 100%   Figure . Patientvs extra-erythrocytic). Young parasites have no pigment and are the most difficult  to ascertain. Malaria pigment is unambiguously present within infected erythrocytes after 30–34 hours of the 48-hour P falciparum life cycle [In a series of 50 autopsies performed on American soldiers dying of clinically defined cerebral malaria during World War II, examination of haematoxylin and eosin-stained slides showed that “the entire capillary network of the brain…was distended with erythrocytes” in 46 cases . In sevefe cycle , and extOf the four measures assessed, the traditional measure, %VP, was most reliable. This measure includes both UPP and PP, and discrepancies in their identification and quantification would be absorbed in the more general measure of %VP. This may have contributed to the enhanced reliability of %VP. The least reliable measures were fibrin and UPP. Smaller studies of fibrin by special stain have demonstrated that CM2 can have ~20% of vessels containing fibrin while CM1 shows ~8% of cases with fibrin . The current validation data appears to have less fibrin than the training data and suggests that studies of fibrin should always include a special stain for fibrin (rather than relying on the appearance of fibrin as stained by haematoxylin and eosin). Similarly, polarized light is a very useful tool for quantifying pigment of any type. Upon expert review, it was clear in the data that UPP were underestimated in the training data by one observer and, thus, this element was used in the repeat CART analysis . For researchers employing this method, the use of a superior light source in combination with extensive training may be required for reliable measurement.The method was used to compare the distribution of all parasites elements in three different areas of the brain. Although the cerebellum appears to be a site of slightly higher overall sequestration, no statistically significant differences in the distribution of parasite elements were observed after correction for multiple testing.A method was developed, validated and applied for determining the intensity and distribution of malaria parasites by life-cycle stage in brain capillaries. Consistent observations are possible, especially for the percentage of vessels parasitized. These methods can be used on the usual formalin-fixed, paraffin-embedded, haematoxylin and eosin-stained tissue, and would allow for more standardized comparisons between different autopsy series. This approach can be used for more detailed studies of the intensity and stage distribution of malaria parasites within and between Malaŵian children dying of falciparum malaria.%VP: Percentage of vessels parasitized; CART: Classification and regression tree; Clin CM: Clinical cerebral malaria by WHO definition; CM1: Children meeting the clinical case definition who have predominantly sequestration only in their brains at autopsy; CM2: Children meeting the clinical case definition who have sequestration and intra and extravascular pathology in their brains at autopsy; CM3: Children meeting the clinical case definition who have no evidence of malarial pathology and another anatomic cause of death; NM Coma: Children in a coma with a non-malarial cause; NM Coma IP: Children in a coma with a non-malaria cause and incidental peripheral parasitemia; Other: Children not meeting definitions of CM or NM Coma; PG: Pigment globules; PP: Pigmented parasites; TPG: Total pigment globules; SMA: Severe malaria anaemia; UPP: Unpigmented parasites.The authors declare that they have no competing interests.DAM designed the study, performed the autopsies, performed the histology experiments, analysed the data, and wrote the manuscript. CV analysed the data and wrote the manuscript. RAC designed the study, performed the autopsies, and performed the histology and electron microscopy experiments. SK and RW performed the autopsies and performed the histology experiments. PBC, NGF and KP performed the histology experiments. KBS, MEM and TET designed the study, recruited the patient population, and wrote the manuscript. All authors read and approved the final manuscript.A demonstration of the counting methods used are presented graphically as a slideshow with notes below each slide explaining the method.Click here for fileA Bland-Altman difference plot for the % of vessels parasitized (%VP) between two observers (NF and DM) across the first 50 cases. The red line marks the mean and the blue lines mark 1 standard deviation.Click here for fileThe graphical correlations between NF and DAM for all parasite elements are demonstrated and show that % of vessels parasites, total parasites, and total pigment globules were well correlate based on Spearman correlations, slope, and p-value.Click here for fileA comparison of the counts for each parasite element made by NF and DAM across all diagnostic categories (non-collapsed) is presented by element. Bonferroni and Sidak corrections for the 70 comparisons sets the p-value at less than or equal 0.00074 for significance. For this level, the counts for fibrin  and pigment globules  were significantly different with NF > DM for both measures.Click here for fileAdditional examples of how different elements are distributed across different diagnoses including total pigment, total parasites, and fibrin based on counts by PC for six brain sites across 28 patients. Data are shown as stacked totals across all sites. In these figures, “non-CM” includes the single CM3 patient in the set of 28.Click here for fileThe quantitative distribution of all parasite elements across the first 50 case by overall measure and by each diagnosis which demonstrates that, overall, fibrin appears to be more common in the parietal lobe ; however, with an appropriate p-value correction (by Bonferroni or Sidak), only values of 0.0014 or less should truly be significant in this data set. At the individual diagnosis level, there were no differences across sites within an individual element.Click here for file


We conducted a surveillance study to determine the leading causes of bloodstream infection in febrile patients seeking treatment at three district hospitals in Pemba Island, Zanzibar, Tanzania, an area with low malaria transmission.All patients above two months of age presenting to hospital with fever were screened, and blood was collected for microbiologic culture and malaria testing. Bacterial sepsis and malaria crude incidence rates were calculated for a one-year period and were adjusted for study participation and diagnostic sensitivity of blood culture.Salmonella Typhi , followed by Streptococcus pneumoniae . The crude bacteremia rate was 6/100,000 but when adjusted for potentially missed cases the rate may be as high as 163/100,000. Crude and adjusted rates for S. Typhi infections and malaria were 4 and 110/100,000 and 4 and 47/100,000, respectively. Twenty three (51%), 22 (49%) and 22 (49%) of the S.Typhi isolates were found to be resistant toward ampicillin, chloramphenicol and cotrimoxazole, respectively. Multidrug resistance (MDR) against the three antimicrobials was detected in 42% of the isolates.Blood culture was performed on 2,209 patients. Among them, 166 (8%) samples yielded bacterial growth; 87 (4%) were considered as likely contaminants; and 79 (4%) as pathogenic bacteria. The most frequent pathogenic bacteria isolated were S. Typhi and S. pneumoniae infections on Pemba Island, Zanzibar. Preventive measures such as vaccination could reduce the febrile disease burden.In the presence of very low malaria incidence we found high rates of  For decades, falciparum malaria has been the leading cause of febrile illness among patients in sub-Saharan Africa presenting to hospitals for treatment. However, during the last ten years, there has been a decline in malaria in many parts of sub-Saharan Africa Following the deployment of artemisinin-based combination therapy starting in late 2003 and the programmatic distribution of long-lasting insecticidal nets from early 2006 in Zanzibar, Tanzania, malaria-associated morbidity and mortality has decreased dramatically The study was conducted according to the principles expressed in the Declaration of Helsinki. Written informed consent was obtained from all study participants or their legal guardians. The Zanzibar Medical Research and Ethics Committee and the International Vaccine Institute - Institutional Review Board in South Korea approved this project.The study was conducted on Pemba, one of the main islands of the Zanzibar archipelago . The totPemba is mainly rural, with an economy based on fishing, seaweed harvesting, and the farming of cloves, coconut, and copra. Primary health care is delivered through 58 primary health care units (PHCU) that provide minor services and can admit one to two inpatients. The units have limited dispensaries but usually have no doctors or laboratory facilities. Patients that require secondary health care are referred to one of the three district hospitals: Chake Chake, Wete, or Mkoani. Additionally, there are the Vitongoji and Micheweni cottage hospitals, which are mainly used during outbreaks of diarrheal diseases.Haemophilus influenzae type b and pneumococcal vaccines are currently not included in the EPI. Typhoid vaccine is not routinely administered in Zanzibar.The Zanzibari Expanded Programme of Immunization (EPI) includes the following vaccines: Bacille Calmette-Guérin (BCG), live oral polio, diphtheria-whole cell pertussis-tetanus-hepatitis B, and the monovalent measles vaccines for children, as well as the supplemental tetanus toxoid vaccine for women of child-bearing age. The study was implemented in phases. Surveillance was started first at Chake Chake District Hospital in March 2009, followed by Mkoani District Hospital in May 2009, and Wete District Hospital in August 2009 . SurveilPatients presenting for care during the study period were screened for eligibility. Subjects above the age of 2 months with a recorded temperature of ≥37.5°C (tympanic thermometer) presenting at the outpatient department (OPD) or subjects above the age of 2 months admitted to a hospital with any history of fever were included in the study. Study staff screened all patients at the OPD during opening hours from 08:00 to 13:00, Monday to Friday. Eligible patients were referred to the study nurse for informed consent, enrolment and blood collection, while waiting to see the hospital health worker who prescribed additional diagnostic tests and treatment, or admitted the patient to the ward. Patients who did not present with a documented temperature of ≥37.5°C but with a history of fever were later enrolled into the study if admitted to the ward. Enrolment of inpatients into the study was conducted in the afternoon after closure of the OPD. Patients admitted during the early evening and night, were screened for eligibility the following morning.Salmonella isolates. The remaining blood samples were used for malaria rapid diagnostic testing  and the preparation of thin and thick blood films. The films were Giemsa-stained and read. At least 300 high power microscopic fields were examined to exclude malaria.We collected 1 to 8 millilitres of blood from children and 9 to 12 millilitres from adults. Immediate bedside testing was done for hemoglobin and glucose . About 1–3 ml of blood from children and 8–10 ml from adults were used to inoculate pediatric and adult BACTEC bottles , respectively, and incubated in an automated blood culture machine . Blood cultures were processed according to standard guidelines Antimicrobial susceptibility testing was performed using the modified Kirby-Bauer disc diffusion method and the mean inhibitory concentration was assessed using Etest. Evaluation was done according to the guidelines of the Clinical and Laboratory Standards Institute Clinical data entered directly into PDAs had real-time error, range and consistency checks, and were transferred to the central database at regular intervals Bacillus spp., Corynebacterium and coagulase-negative Staphylococci. Malaria was defined as fever with a positive blood film and/or positive rapid diagnostic test (RDT).The following definitions were applied during the analysis. Bacteremia was defined as fever with isolation of pathogenic bacteria from blood culture. Contaminants were defined as non-pathogenic bacteria most probably derived from the skin during blood collection and included In order to avoid seasonal confounding in the incidence rates, we used data from January to December 2010 only. Incidence rates were calculated per 100,000 persons and by age group: ≤5, >5–15 and >15 years old. The total and age-specific number of cases was used as the numerator and the respective population for 2010 projected from the 2002 census All analyses were performed using Microsoft Excel spreadsheets and Stata version 10 .Plasmodium falciparum), whereas 28 (1%) had malaria  with a negative blood culture .We assessed the bacteremia cases by hospital . Of the S. Typhi was found to be the leading pathogen in all age groups, causing 10/29 (35%), 12/16 (75%) and 24/34 (71%) of bacteremia, respectively and 46 cases (58%) overall. The second most frequently isolated pathogen was Streptococcus pneumoniae, which was responsible for 12 (15%) bacteremia cases overall; and 8/29 (28%) among those ≤5 years old, 3/16 (19%) among those 5 to 15 years of age and 1/34 (3%) among those >15 years. Other commonly isolated bacterial pathogens were Escherichia coli, Staphylococcus aureus, and Haemophilus influenzae type b (Hib).Of the 2,209 patients included in the analysis, 637 (29%) were ≤5 years old, 490 (22%) were >5 to 15 years old, and 1,082 (49%) were >15 years old . The patWe calculated the crude and adjusted incidence rates for bacteremia  and falcS. Typhi, had a crude incidence rate of 4/100,000 (95%CI: 3 to 6) and an adjusted rate of 110/100,000 (95%CI: 102 to 120). The age-specific incidence rates of typhoid fever increased with age  and 1/100,000 (95%CI: 0.2 to 2) if only slide-positive cases were included in the analysis . AdjustmS.Typhi isolates  against the three first-line antimicrobials was detected in 19/45 (42%) isolates. Out of these 19 MDR isolates, four (21%) were resistant to nalidixic acid, but none of them was resistant to ciprofloxacin.Antibiotic susceptibility was assessed for 45/46 (98%) isolates  and for isolates . Twenty S. Typhi to be the leading pathogen in adults and children presenting to hospitals with severe febrile illness in an area of low malaria transmission. Our adjusted rate of 110 typhoid fever cases per 100,000 population falls into the category of “high typhoid incidence area ” based on the classification by Crump et al.S. pneumoniae was the second most frequently isolated pathogen in all age groups.We found that Salmonella. Falciparum malaria is associated with invasive non-typhoidal Salmonellosis and also likely accounts for these differences Incidence data on community-acquired bacteremia from this region is sparse. A study from 2006 from western Kenya reports incidence rates of bacteremia among children below the age of 2 and below 5 years to be 1,741 and 909 per 100,000 children, respectively et al.S. enterica . Fifty eight percent of these were non-typhoidal Salmonella, 34% were S. Typhi, and 8% were unspecified S. enterica isolates. A recent study by Crump et al found S.Typhi the leading pathogen among HIV negative children in the Moshi area with 6 out of 341 patients Reddy We found significant differences in the isolation rate of pathogenic bacteria among hospitals. Potential explanations for this phenomenon might be differences in health-seeking behaviour. The differences were not due to outbreaks since the cases were rather evenly distributed over the entire study period.S. Typhi, all isolates were found to be susceptible to at least one locally available antibiotic. About half of the Salmonella Typhi isolates were resistant to at least one of the frequently used drugs: ampicillin, chloramphenicol, and trimethoprim-sulfadoxine. More than 40% showed resistance toward all three drugs (multi-drug resistance). These are considerably higher resistance rates than those reported between 1995 and 2008 With the exception of The current routine treatment for inpatients with suspected bacteremia is ampicillin and gentamicin. If this fails and subject to availability ceftriaxone is given. For outpatients ciprofloxacin is routinely given if bacteremia is suspected. According to our data this strategy seems appropriate especially since full susceptibility towards ceftriaxone was found for S.Typhi, E.coli and Hib.This study had several limitations. Firstly, incidence calculations were adjusted for health-seeking behaviour based on findings from four villages around Chake Chake District Hospital only . Variations in health-seeking behaviour by geographic distance to the hospital, sex, or age were not taken into account. It is possible that a larger percentage than estimated make use of the participating health care facilities during a prolonged course of disease, if not as the first choice, then as a later alternative. Secondly, the study period was brief and no changes in disease trends could not be detected. Thirdly, only single blood cultures were collected. Repeated sample collection and multiple cultures from each patient might have increased the overall positivity rate S. pneumoniae among children on Pemba Island, Zanzibar. The data indicate the need to consider preventive strategies against these diseases to further decrease the burden of severe febrile illness. These include long-term improvements in education, hygiene behaviour such as hand-washing, water supply sanitation to address the high S.Typhi incidence. In addition vaccination programmes against typhoid fever and the introduction of the pneumococcal vaccine in the EPI program deserves consideration.In summary, in the presence of very low malaria incidence we found high rates of typhoid fever in all age groups and