Plasmodium vivax hypnozoites and killing gametocytes of Plasmodium falciparum. However, this drug causes haemolysis in individuals who are glucose-6-phosphate (G6PD) deficient. It is important to map G6PD deficiency and malaria distribution in Pakistan to design an effective malaria eradication regimen. Frequency of G6PD deficiency (G6PDd) in malaria patients has not been reported from Pakistan in any meaningful way. The purpose of this study was to evaluate the frequency of G6PD c.563C>T (G6PD Mediterranean) in male individuals with and without falciparum malaria.Pakistan has an estimated annual burden of 1.5 million malaria cases. The current situation calls for an effective malaria control and eradication programme in this country. Currently, primaquine is an attractive option for eliminating reservoirs of P. falciparum histidine-rich protein 2. Parasitaemia was also computed. DNA samples were tested for G6PD c.563C>T mutation through PCR–RFLP according to the previously defined protocol and its allelic frequency was computed.Two hundred and ten archived DNA samples from males  were utilized in this study. Healthy blood donors were selected based on stringent pre-defined criteria. Patients were confirmed for malaria parasites on microscopy and or immune chromatographic assay detecting G6PD c.563C>T was observed in four of 110 patients with falciparum malaria and in two of 100 healthy donors. Mean (± SD) haemoglobin, median (IQR) platelet and median (IQR) parasite count in G6PD-deficient malaria-patients were 8.9 ± 0.9 g/dL, 124 × 109/L  and 57,920/μL of blood  respectively.G6PD 563c.C>T was 0.0285 detected in 6 of 210 X-chromosomes in Southern Pakistan. Frequency for this G6PD allele was 0.0364 in malaria-patients and 0.0200 in healthy individuals. Large studies including females are needed to elucidate the true burden of G6PDd in malaria-endemic areas. The information will enable local health policy makers to design effective strategies for eliminating malaria form this region.Cumulative allelic frequency for  Plasmodium vivax is endemic and more common (88%) in this country, while malaria with Plasmodium falciparum is seen only during rainy seasons or post rain and accounts for 12% of the malaria burden [P. vivax hypnozoites [P. falciparum gametocytes [With an estimated load of 1.5 million malaria cases each year, Pakistan is among seven countries of the WHO Eastern Mediterranean Region sharing 95% of the total regional malaria burden [nozoites  and P. fetocytes . HoweverG6PD gene is located on the X-chromosome, the prevalence of G6PD in males is higher than that of females. Interestingly, global distribution of G6PD deficiency (G6PDd) matches that of past and present malaria [G6PD A-type offers relative protection against falciparum malaria in hemizygous males and to some extent in heterozygous females [G6PD Mediterranean (G6PD-Med)—a variant common in Asian countries confers immunity against vivax malaria [G6PD is a pentose pathway enzyme which catalyzes the rate limiting step in the reduction of nicotine amide adenine dinucleotide phosphate (NADP). Reduced NADP is critical for eradicating free radicals from the cells and maintaining their viability. Therefore, generally asymptomatic, G6PD-deficient individuals are at risk of drug-, food- or infection-induced acute haemolytic anaemia. Because the  malaria , suggest malaria  in hemizG6PD-Med, is the most common G6PD variant in Pakistan [G6PD-Med [Previous work has shown that G6PD deficiency (G6PDd) is widely prevalent in Northern Pakistan with a frequency ranging from 2 to 8% in various ethnic groups [Pakistan . Ashley G6PD-Med . This inG6PD-Med protect hemizygous males and heterozygous or homozygous females from P. falciparum? To respond to these basic questions, large-scale studies are needed. The present study was aimed in determining allelic frequency of G6PD c.563C>T in male individuals with or without malaria by using archived DNA samples. This small-scale study is anticipated to become a basis for larger studies in evaluating the true burden of G6PDd before planning mass primaquine therapy.There is limited information for G6PD-malaria interaction in Pakistan raising several research questions: What is the prevalence of G6PDd in Southern Pakistan? Does This was a descriptive study conducted in 2013 at a tertiary care academic institute, Aga Khan University Hospital (AKUH), Karachi, Pakistan. Karachi is the largest city of Pakistan and is the residence to several ethno-lingual groups from all over the country. Muhajirs (emigrants from India) constitute 50% of its population, while Pashtuns are the second largest group. Other ethnic groups include Sindhis, Punjabis, Bengalis, Anglo-Indians, Parsi and Catholics. AKUH because of its location caters mostly Karachites. Therefore, it can be anticipated that ethnic background of individuals visiting AKUH or its blood bank will be homogenous. This is more rightly so, as blood bank works on the principle of exchange donations thereby prompting relatives of admitted patients to donate blood. Approximately 40,000 subjects donate blood annually at AKUH blood bank of which 99% are male donors. The total number of admissions with malaria in AKUH between January 2009 and December 2011 was 365 [Two hundred and ten archived DNA samples were utilized in the study. This included 110 males who were infected with falciparum malaria during 2006–2007 and 100 healthy males who donated blood at AKUH during the same time period. Random sample identification technique was used to select patients from a large pool of archived DNAs. The focus was to determine G6PDd allelic frequency in males who were infected with falciparum malaria  for which we had limited archived samples in AKUH DNA bank. EDTA blood sample was collected at the time patient presented with malaria, DNA extracted and archived at − 80 °C in the research laboratory for future usage. Healthy blood donors were selected based on stringent pre-defined criteria for blood donation such as no history of malaria in past 3 years. G6PD deficiency is an x-linked disorder and mainly affects males, therefore, only males were included in this study. Moreover, national data for G6PDd was available for males predominantly. In addition, there is a preponderance of male blood donors in Pakistan—the control group in this study. Female patients and all other patients suffering from vivax malaria were excluded.P. falciparum histidine rich protein2 (HRP2). Briefly, thick blood films were air dried and dipped in water to remove haemolyzed red cells. Slides were then stained with 4–5% Giemsa with phosphate buffer saline (pH 7.2) for 25 min and then rinsed with tap water. Thick films were examined microscopically under high power field (100× oil immersion lens) for parasitized erythrocytes against 200 white blood cells. Parasite density was estimated on thick film assuming a standard value of 8000 white blood cells per microlitre of blood. Parasitaemia was calculated as follows:For blood donors, ICT malaria test was used for screening malaria. Patients were routinely diagnosed on microscopy (of thick and thin films) and or immune chromatographic assay (ICT) for Number of parasites = Total parasite counted × 200 white cells/No of white cells/µL of blood. Thin peripheral smears were stained with Leishman’s stain and reviewed at 40× and 100× objectives.Medical charts were reviewed for various clinical (respiratory and nervous system manifestations) and laboratory features  were retrieved from the computerized laboratory data system. Duration of hospital stay was also determined. Patients were divided into paediatric and adult groups and the cut off was ≤ 17 and > 17 years, respectively, as per institutional policy.G6PD c.563C>T mutation through previously defined protocol [G6PD Chatham were not studied as the study was focused on determining relation between G6PD-Med (predominant and severe form) and P. falciparum .Archived DNA samples from malaria patients (n = 110) and blood donors (n = 100) were tested for protocol . Other rEthical approval for this study was granted by The Aga Khan University Ethical Review Committee (#509-Pat/ERC-06). Informed consent was taken from blood donors and malaria patients for genotyping. Re-coding was done to assure anonymity of the subjects.G6PD variant. Descriptive analysis (mean ± SD with range) was computed for quantitative data with normal distribution and median and inter-quartile range (IQR) for skewed data. Laboratory parameters were compared with respect to age group and the threshold of significance was below 0.05. G6PD allele frequency was computed as number of affected males divided by total number of males.Data was entered in Microsoft Excel sheet and transferred to SPSS version 22  for statistical analysis. Frequency was calculated for age and One hundred and ten malaria-infected males with a mean age of 27.2 ± 17.5 years were studied. Twenty-nine % of patients were children below 17 years of age. The blood donors were young males of mean age 27 ± 7.0 years (range 17–52).Clinical details were available for only 48 malaria patients (7 children and 41 adults) who were hospitalized. Significant clinical findings included fever , splenomegaly  and pulmonary oedema (n = 3). Six adults demonstrated complications like cerebral malaria (n = 3), respiratory failure (n = 2) and renal failure (n = 1). Mean stay (in days) in hospital and intensive care was 4.38 ± 4.33 and 0.89 ± 2.99 days respectively. No mortality was observed in any patient. Treatment included artemisinin (n = 4), doxycycline (n = 8), quinine (n = 4), and combination of anti-malarial in rest of the patients. Blood donors recruited in the study had no history of malaria in past 3 years. Two patients carrying G6PD mutation did not show complicated malaria and received artemisinin plus doxycycline and quinine.Laboratory details were available for all malaria patients n = 110). Eighty-two percent patients were anaemic, 14.5% were severely anaemic (with haemoglobin < 7 g/dL), 66% patients had thrombocytopaenia while 12% were leucopaenic. Twenty patients had haemoglobinuria as reported in urine analysis. Laboratory parameters with respect to age group are summarized in Table 10. EightG6PD 563c.C>T was detected in four of 110 patients with falciparum malaria and in two of 100 healthy blood donors. Cumulative allelic frequency for G6PD 563c.C>T was 0.0273 detected in 6 of 220 X-chromosomes (Table G6PD 563c.C>T. Mean (± SD) haemoglobin, median (IQR) platelet and median (IQR) parasite count in G6PD-deficient malaria-patients were 8.9 ± 0.9 g/dL, 124 × 109/L  and 57,920/μL of blood , respectively.G6PD-Med in subjects infected with malaria in Southern Pakistan and 2.8% in all tested individuals. This is lower than average allelic frequency reports for G6PD deficiency from various regions of Pakistan. But considering that only one G6PD mutation (G6PD 563c.C>T) was looked for, we would have captured 80% of the G6PDd prevalence. Other G6PD variants reported from Pakistan were G6PD Orissa and Chatham [This study showed an allelic frequency of 3.6% for  Chatham , for whi Chatham . Literat Chatham –27 with  Chatham . The datP. falciparum transmission globally through mathematical modeling [P. falciparum. This was observed in the current study as well, as four of 110 patients with falciparum malaria had G6PD 563c.C>T mutation and were not immune to malaria. Moreover, the G6PDd malaria patients had high parasite burden of 152,821.00 ± 259,297.01/μL in this study. In contrast, Guindo et al. reported protective effect of G6PD A-type in African males against falciparum infection [G6PD 563c.C>T in Pakistani population that may have different effect on P. falciparum survival than that observed in G6PD A-African population. More studies are needed on the immunity (if any) conferred by G6PD 563c.C>T against P. falciparum in hemizygous males and heterozygous females.Gething et al. in 2011 estimated modeling . Accordimodeling . Khyber modeling . There imodeling . InteresG6PD 563c.C>T is highly prevalent in West Asia including Pakistan [Pakistan . This muPakistan . The effG6PD 563c.C>T variance in Southern Pakistan demonstrating presence of this variant in patients suffering from malaria. The study had certain limitations as mutational analysis in females and in patients suffering from vivax malaria was not done. G6PD assay was not done hence the true prevalence of G6PD deficiency was underestimated. Moreover, other G6PD mutations except G6PD 563c.C>T were not identified. However the study did provide a snapshot for the presence of severe form of G6PD deficiency in Southern Pakistan.The study provides a database for G6PD 563c.C>T was 0.0273 detected in 6 of 220 X-chromosomes. The allele frequency is in line with previously reported data from Pakistan. This study advocates mandatory need for G6PD testing prior to anti-malarial intervention with primaquine therapy. Escalating doses of primaquine in patients with G6PD deficiency needs to be addressed in future studies.Cumulative allelic frequency for


The current roll-out of rapid diagnostic tests (RDTs) in many endemic countries has resulted in the reporting of fewer cases of malaria-attributed illnesses. However, lack of knowledge of the prevalence of other febrile illnesses and affordable diagnostic tests means that febrile patients are not managed optimally. This study assessed the prevalence of commonly treatable or preventable febrile illnesses in children between 6 months and 15 years using rapid diagnostic tests at the point-of-care.Febrile children were enrolled between February-April 2014 at a health facility after obtaining informed consent from parent. Eligible participants were aged 6 months-15 years with a history of fever in the last 24 h or axillary temperature ≥38 °C at consultation. All participants were tested using RDTs for malaria, typhoid, toxoplasmosis and rubella. Malaria parasites were further identified by microscopy and PCR. Clinical and household characteristics were recorded and association with pathogens determined.p = 0. 029) as well as drinking water and presence of typhoid (p = 0.047). No association was observed between type of housing and malaria, or toxoplasmosis and raising cats.Of the 315 children enrolled, the mean age was 5.8 ± 3.8 years. Stomach pain (41.2 %) was the most reported symptom. Prior to attending the health facility, 70.8 % had taken antipyretics, 27.9 % antimalarials, 11.4 % antibiotics and 13.3 % antifungal drugs. Among 315 children with fever, based on RDTs, 56.8 % were infected with malaria, 4.4 % with typhoid, 3.2 % with acute toxoplasmosis, and 1.3 % with rubella . All non-malarial infections were co-infections and approximately 30 % of the fever cases went un-diagnosed. Malaria prevalence by microscopy and PCR was 43.4 and 70.2 % respectively. The sensitivity and specificity of RDTs for the diagnosis of malaria were 75.98 and 100 % respectively, with 0.73 measurement agreement between RDTs and microscopy while that of RDT and PCR were 81 and 100 % respectively with a K value of 0.72. The use of Insecticide Treated Bednets was 44 %. There was a significant association between ITN non-usage and malaria  contains supplementary material, which is available to authorized users. Plasmodium falciparum infections. Serious consideration of other etiologies may not occur unless there is no clinical response to antimalarials treatment.Fever is the most common symptom of infectious diseases in tropical countries. A wide range of treatable or preventable pathogens are known to cause fever in patients who present with malaria-like symptoms, but do not have malaria . MalariaMany malaria endemic countries including Cameroon have adopted the WHO evidence -based guidelines for effective case management of malaria and rapid diagnostic tests (RDTs) have been presented as a means to realize this key strategy . The recDiagnosis of non-malaria febrile illness in resource limited settings remains challenging . With maWith the successful contribution of RDTs as a key strategy in evidence-based malaria case management, RDTs, designed for peripheral health facilities have been developed or are being developed for other tropical infections including typhoid, toxoplasmosis, to improve patient and epidemiological surveillance. Most RDTs for non-malarial tropical infections are reported to rely on the detection of host antibodies against a single infectious agent and the sensitivity and specificity of host-antibody detection tests are both inherently limited . HoweverA number of infectious diseases such as typhoid fever, rickettsial and arboviral infections have been recognized among febrile adult patients attending health facilities in Cameroon –21 whileThe study was carried out in Nkolbison Health District, Yaoundé from February-April 2014. Nkolbisson is located in Mfoundi Division of the Centre Region of Cameroon, about 6 km from Yaoundé City Centre. Yaoundé is the capital of Cameroon with an estimated population of 2.5 million people. Malaria transmission in Yaoundé is perennial with two main seasons: the long wet season from February to November (with more intense rains between September and November) and a short dry season from June to July and December to January .The target population was children between 6 months-15 years who came for outpatient consultation at the health facility. Children of both sexes were eligible if they were within the age limit, had a history of fever in the preceding 24 h or axillary temperature ≥38 °C on consultation, and the parent /guardian gave a written informed consent for his/her child to participate in the study. Since information on the prevalence of toxoplasmosis, rubella and typhoid for children with fever was not known, sample size was calculated based on malaria morbidity in children (38 %) as indicated in the National Malaria Control Program (NMCP) report in 2011 [For all participants who met the inclusion criteria at the outpatient department of the health facility, a baseline questionnaire was administered to collect information on participants and the characteristics of their households. Data included: demographic data, signs and symptoms, type of medication taken prior to health facility visit, type of housing, main source of drinking water, ownership and usage of insecticide treated bed nets (ITN), and type of domestic animal in household.~ 20 μl spotted on filter paper (Whatman No.3) for DNA extraction and molecular studies. The rest of the blood was centrifuged and the plasma used to perform RDTs for typhoid, toxoplasmosis and rubella. Plasma was also aliquoted into cryogen vials, transported on ice packs to the Research Laboratory at the Biotechnology Centre and stored at -20 °C.A total of 2 ml of whole blood was collected from each participant. Of these, half of the blood was used to perform the malaria RDT on site, prepare thick blood films for microscopy, determine hemoglobin level by URIT 3200 automated hematology analyzer  and Pf/Pan antigen rapid test is a qualitative and rapid immuno-chromatographic test for the differential diagnosis of P. falciparum histidine rich protein II (P.f HRP-II) and lactate dehydrogenase (pLDH) common to P. falciparum, P.malaria, P.ovale and P.vivax (Pan). Briefly, 5 μl of whole blood was placed in the sample well, 4 drops of diluent added and results read in 15 min. The test was considered positive if there was a red line on the control (C) and test line  or C and two test lines  or C and test line  and negative when only the C line was observed.The Malaria Ag Pf/Pan RDT  was used for malaria diagnosis. The Malaria Ag OnSite Typhoid IgG/IgM Combo rapid test . The OnSite Typhoid IgG/IgM Combo rapid test cassette is a lateral flow chromatographic immunoassay that can detect and differentiatiate of IgG and IgM antibodies to -Salmonella typhi and S. paratyphi in human blood. Briefly, one drop of plasma (50 μl) was added to the sample well, immediately followed by one drop of sample diluent. Results were read in 15 min. The appearance of a burgundy colored band in the C (control) line and in the M or G lines was considered to be positive for IgM and IgG antibodies, respectively. Meanwhile the absence of a coloured band in both the G and M lines, but present in the C line was considered negative.Diagnosis of typhoid was done using the OnSite Toxo IgG/IgM rapid test . The OnSiteToxoIgG/IgM rapid test simultaneously detects and differentiates of IgG and IgM anti-Toxoplasma gondii antibodies in human serum or plasma. A total of 50 μl of plasma was placed into the sample well and a drop of sample diluent added and the results read within 15 min. The test was negative if only the control band (C band) developed and no burgundy colour observed in the both Test bands (T1 and T2) indicating the absence of anti- T. gondii antibodies in the specimen. Meanwhile, a positive test was indicated by the presence of a C band and a T1 band (IgM anti-T. gondii) or the presence of C and a T2 band (IgG anti-T.gondii) or the presence of both C band, T1 and T2 bands.Diagnosis of toxoplasmosis was done using the Diagnosis of rubella was carried out using the SD BIOLINE Rubella IgG/IgM rapid test . The SD BIOLINE Rubella IgGIgM kit is intended to indicate the immune status or confirm recent rubella infection. Five microlitres (5 μl) of sample were placed into the sample well, 4 drops of diluent added and results read within 30 min. A test was positive if two pink lines C (Control) and G (IgG) or C and M (IgM) or three pink lines C, G and M developed in the result window. A negative test was indicated by one pink line C in the result window.Thick films stained with 10 % Giemsa were used for definitive parasite counts; 200 high power fields were screened before a slide was declared negative. The number of parasites per 200 leucocytes was recorded and converted into parasite density per μl by assuming an average white blood cell count of 8000/μl. The mean of 2 slide readings from 2 independent readers was performed and discrepancies greater than 10 % were performed by a third reader .DNA was extracted from dried blood spot on filter papers from each participant by chelex boiling method as described by . The 18sData were analysed using SPSS version 16.0 statistical software. Descriptive statistics were used to determine the prevalence of each of the pathogens and past infections. The sensitivity, specificity, positive and negative predictive values of RDT versus microscopy and PCR for malaria diagnosis were determined using Epi Info version 7 and the 2- Tailed P exact Fisher test used for comparisons. Crude odds ratio (ORs) with their 95 % confidence intervals (CI) were estimated for association between household characteristics , P.ovale 0.63 % (2/315), P. malariae 0.63 % (2/315), P. falciparum + P.ovale 5.4 % (17/315), P. falciparum + P. malariae 0. 32 % (1/315) and 29.84 % (94/315) were negative.Prevalence of malaria, typhoid IgM, toxoplasmosis IgM and rubella IgM antibodies by RDTs was 56.8, 4.4, 3.2 and 1.3 % respectively  compared to those who did not use an ITN (Table p = 0.072), poorly constructed house with blocks , poorly constructed house with planks  compared to those who lived in modern houses. A significant association between main source of drinking water and typhoid was observed for participants who had a borehole or well  compared to those with piped water into the dwelling while no significant association was found between acute toxoplasmosis and raising cats (p = 0.060).An odds ratio analysis showed a significant association between ITN usage and In this study, we determined the causes of commonly treatable or preventable febrile illnesses in children between 6 months and 15 years using rapid diagnostic tests at the point-of-care. All the 315 children in this study presented with fever and other symptoms that are common to malaria and other febrile illnesses like typhoid, toxoplasmosis and rubella thus confirming the need for a differential diagnosis of febrile illnesses as reported in many other malaria endemic countries –21. SelfP. falciparum still remains the most prevalent malaria parasite species. This shows that malaria prevalence is still high amongst children in Cameroon and remains the main cause of fevers in this population despite the introduction of current control efforts such as the use of insecticide treated bednets (ITNs). Though RDTs were more sensitive compared to microscopy, and they were significantly less sensitive compared to PCR  IgM antibodies seroprevalence, as diagnosed by RDTs, was 4.4 % and these patients presented with malaria—like symptoms and were also positive for malaria. These results were similar to the reports of Nsutebu et al., [, [p = 0.047). Public health measures such as improved personal hygiene and intensive community health education campaign [Typhoid and malaria share social circumstances which are important for their transmission, and so individuals in areas endemic for both diseases are at substantial risk of contracting both these diseases, either concurrently or an acute infection superimposed on a chronic one , 39, 40. et al.,  who obse et al.,  who repo al., [,  that typToxoplasmosis infection in immunocompetent adults and children are usually asymptomatic or have symptoms that spontaneously resolve such as fever, malaise, and lymphadenopathy indicating a symptomless latent infection [T.gondii) IgM antibodies of 3.4 % and occurred as co-infection with malaria. Most of the toxoplasmosis infection observed in this study might have been congenital given the high prevalence previously reported in pregnant women [nfection . A repornfection . Febrilent women  in a simnt women . HoweverRubella is a mild disease in children and adults, but can cause devastating problems if it infects the fetus, especially when the infection occurs during the first weeks of pregnancy , 44. TheThis study also has some limitations. First, the methods used in this study were unable to determine the cause of fever in approximately 30 % of the study population that were negative for all the four pathogens investigated. Investigations of other pathogens of viral and bacterial origin and biomarkers could provide additional useful diagnostic clues that may help to refine decision-making. Moreover, the RDTs for typhoid, toxoplasmosis and rubella maybe non-specific hence confirmation with more specific and sensitive tests such as cultures or PCR when available may be useful. Secondly, it is a health-facility-based study and it did not include patients within the community. Also, the influence of other factors such as the economic status of the family, education and the nutritional status of the children were not investigated.Although the prevalence of typhoid, toxoplasmosis and rubella was low in this population, it will still be important to consider a differential diagnosis of these febrile illnesses so as to reduce overdiagnosis of malaria and overprescription of antimalarials.Though malaria still remains the major cause of fever in children, using RDTs of other treatable febrile illnesses like typhoid and toxoplasmosis could facilitate the optimal management of febrile illnesses at the point- of- care. Results from RDTs also provide useful information to health-workers on how to manage febrile patients at the point of care either when malaria is negative or exists as a co-infection with other infectious diseases such as typhoid and toxoplasmosis


Plasmodium falciparum infections and reduce the human parasite reservoir.Mass drug administration (MDA) using dihydroartemisinin plus piperaquine (DHAp) represents a potential strategy to clear A cluster-randomized controlled trial in Southern Province, Zambia, was used to assess the short-term impact of 2 rounds of community-wide MDA and household-level  MDA with DHAp compared with no mass treatment. Study end points included parasite prevalence in children, infection incidence, and confirmed malaria case incidence.P = .04). No difference between treatment groups was observed in areas of high transmission. The 5-month cumulative infection incidence was 70% lower  and 58% lower  after MDA compared with control in lower- and higher-transmission areas, respectively. No significant impact of focal MDA was observed for any end point.All end points significantly decreased after intervention, irrespective of treatment group. Parasite prevalence from 7.71% at baseline to 0.54% after MDA in lower-transmission areas, resulting in an 87% reduction compared with control (adjusted odds ratio, 0.13; 95% confidence interval, .02–.92;Two rounds of MDA with DHAp rapidly reduced infection prevalence, infection incidence, and confirmed case incidence rates, especially in low-transmission areas.NCT02329301. Over the past decade, Zambia has successfully scaled up the World Health Organization–recommended malaria control interventions  and is considering alternative strategies to further reduce the malaria burden . AlthougMass testing and treatment interventions, wherein individuals are tested with a rapid diagnostic test (RDT) and treated if results are positive, have had limited impact on malaria in Southern Zambia and elsewhere –7, primaThe primary goal of using 2 rounds of MDA or fMDA with DHAp in Zambia is to reduce the malaria parasite infection prevalence and incidence to preelimination levels during peak transmission in low-transmission areas  and to hThe full protocol for this trial has been published elsewhere . EthicalA cluster-randomized controlled trial (CRCT) was used to evaluate the impact of the mass treatment interventions on study end points. The trial area was stratified into higher- and lower-transmission strata above and below 10% parasite prevalence at randomization. The study was conducted in Southern Province, Zambia along Lake Kariba in 60 health facility catchment areas (HFCAs) in 10 districts  . HousehoPlasmodium falciparum infections during the mass treatment rounds. All individuals meeting the inclusion criteria to receive DHAp were offered an age-appropriate 3-day course of DHAp based on manufacturer's recommendations and national treatment guidelines [DHAp  was used presumptively to treat idelines . The firAll individuals in intervention clusters were tested for parasite infections using an RDT  during each of the 2 house-to-house mass treatment rounds. MDA consisted of offering all eligible individuals DHAp, irrespective of RDT result . fMDA consisted of offering DHAp to all eligible individuals who resided in a household where anyone tested positive by RDT . The control group received the standard of care, described above, but did not receive any mass treatment intervention.Children <3 months old and pregnant women in their first trimester were excluded from receiving DHAp, according to the manufacturer's recommendations; they were instead offered the appropriate dose of antimalarial treatment according to Zambian national policy if RDT positive . All individuals with suspected severe malaria or other severe illness were referred to the nearest health facility and omitted from the study.The primary end point was malaria parasite infection prevalence among children ≥3 months to <6 years old, defined as the proportion of children with a malaria parasite infection by RDT. Secondary end points included the cumulative infection incidence rate among all persons ≥3 months old , and the confirmed malaria case incidence rate .Malaria parasite infection prevalence in children was measured by a simple random sample of households during the high-transmission season (April–May) before the mass treatment rounds in 2014 (baseline) and again after the mass treatment rounds in 2015 (follow-up). A sample size of 2820 children at each survey round was required to detect a 50% reduction in infection prevalence with 80% power taking into account the cluster randomization, as described elsewhere . RDTs anThe cumulative infection incidence rate was measured in individuals enrolled in a prospective cohort followed from December 2014 through the end of May 2015 . A target sample size of 2250 individuals was sought to detect a 50% reduction in infection incidence between either mass treatment group and control, with 80% statistical power taking into account the cluster randomization, as described elsewhere . Cohort Routine data from the health management information system on monthly laboratory-confirmed outpatient malaria cases were ascertained from all 60 healthcare facilities in the study area from January 2011 onward. Confirmed case counts were standardized by the estimated midyear populations of each HFCA to obtain the incidence per 1000 population.The CRCT study design was accounted for by including the cluster (HFCA) as a random effect in all analyses. All analyses were intention-to-treat analyses wherein all individuals were assumed to receive the treatment assigned to their HFCA at randomization. The treatment effect between the MDA/fMDA and control group for malaria infection prevalence in children was estimated for the 2015 follow-up (posttest) survey time point, using a crude odds ratio (OR) in a bivariate logistic regression model. A secondary model, defined a priori, adjusted for child age (in years), sex, household wealth, rainfall, the enhanced vegetation index, household elevation, and household protection vector control.Crude incidence rate ratios (IRRs) were used to estimate the treatment effect of the MDA/fMDA on the cumulative infection incidence, compared with the control group, using a negative binomial model. Individuals present at enrollment that completed ≥3 months of follow-up were included in the analysis.The treatment effect of MDA/fMDA on the confirmed malaria case incidence, compared with the control group, was estimated using a negative binomial model, standardized by midyear HFCA population. The model controlled for previous month's cases, calendar month, and anomalies in monthly rainfall and enhanced vegetation index. The baseline preintervention period was January–May 2013 and January–May 2014, and the follow-up postintervention period was January–May 2015. Because there were significant differences across treatment groups in the monthly confirmed malaria case incidence during the baseline period, a difference-in-differences model was used to account for baseline differences.Rounds 1 and 2 of the MDA and fMDA were successfully implemented in December 2014 and February–March 2015, respectively. Based on the 2015 follow-up survey, household coverage by MDA teams was 88.1% and 72.0% for rounds 1 and 2, respectively, and 62.5% and 54.0% for fMDA, respectively (Supplementary Table 1). The rate of adherence to a full course of DHAp was >85%, and the rate of refusal to partake in the study was very low across treatment rounds at <1%. Monthly total rainfall amounts during the study period were similar between treatment groups .P = .09), and a statistically significant 87% relative reduction  after accounting for confounding factors. No other significant differences were observed.The baseline parasite survey conducted from April–May 2014 before the mass treatment interventions showed child infection prevalence, child and household demographics, treatment seeking for fevers, intervention coverage, and climate to be similar across treatment groups (Tables P = .14) (Table P = .046). No other significant differences were observed.A total of 2138 individuals were enrolled into the cohort from 1 December 2014 to 16 January 2015 and followed up through 13 June 2015 . No other significant differences were observed.Large declines in the monthly confirmed malaria case incidence rates were observed after the mass treatment rounds, including in the control group Table . Among HWe evaluated the short-term impact of 2 rounds of MDA or fMDA with DHAp against a control of no mass treatment in Southern Province, Zambia, in the context of strong prevention and surveillance efforts. To our knowledge this is the first CRCT assessing the impact of mass treatment interventions with DHAp in an African setting.Large declines in study end points were observed in both intervention and control areas. Despite this, results during the first 5 months of the trial show 2 rounds of MDA with DHAp to have substantial impact on study end points in this setting. In areas of lower transmission, MDA was shown to reduce peak season child parasite infection prevalence from 8% before to <1% after MDA (87% decline). Individuals receiving MDA experienced a 3-fold decrease in cumulative infection incidence in lower-transmission areas (nonsignificant) and a significant 2-fold decrease in higher-transmission areas. These results are supported by monthly confirmed case incidence data derived from the health management information system.Results from the trial were largely consistent across study end points. First, fMDA showed consistently less impact than MDA, with no results reaching statistical significance. Second, across all study end points, the effect size of 2 rounds of MDA was larger in lower-transmission than in higher-transmission areas.It is likely that RDTs used during fMDA for treatment decisions still missed a substantial proportion of individuals with low-density parasite infections , 10. FurAlthough we identified many reports describing the short-term success of MDA campaigns under program settings , 18–24, We observed large decreases in malaria infection prevalence in children (by 73%) and confirmed malaria case incidence by 43%) in control areas that coincided with mass treatment implemented in neighboring intervention areas. As a result, the expected detectable differences in these end points for comparing the mass treatment interventions to control was much lower than expected, resulting in lower statistical power than expected to detect significant differences . It is u3% in conResults of this trial should be interpreted in light of several limitations. First, the follow-up period of 5 months is insufficient to assess long-term trends in the malaria burden after mass treatment. However, the short follow-up period does allow measurement of short-term impact of mass treatment at driving the malaria burden down to preelimination levels. Two additional rounds of MDA and fMDA with DHAp were conducted in October 2015 and February 2016; we are continuing to collect data on primary end points and will report longer-term results once available. Second, because of the relatively small number of clusters (10 HFCAs in each treatment group in each transmission stratum), there was the potential for imbalance across treatment groups. However, all analyses of study end points also included models with potential confounders, stipulated a priori in the protocol [Third, we performed an intention-to-treat analysis; misclassification of the true exposure between MDA, fMDA, and control households owing to contamination across HFCA borders was possible. To mitigate misclassification and contamination, households within a 3-km buffer of HFCA boarders were excluded from the sampling frame for malaria parasite prevalence and cumulative infection incidence. Misclassification of HFCA into lower- and higher-transmission strata above and below 10% parasite prevalence was also possible. Fourth, the cumulative infection incidence analysis used RDTs. Although slides were collected and read for the cohort, positivity among slides was only a fraction of RDT positivity, probably a result of poor slide preparation among the 16 000 samples collected in the field. It is possible that RDTs were prone to yield more false-positive results than slides , especiaAlthough there is no clear evidence linking MDA to antimalarial drug resistance , the potIn conclusion, 2 rounds of MDA had a substantial impact on malaria infection prevalence, cumulative infection incidence, and confirmed case incidence rates, especially in lower-transmission areas. It is important to highlight that the trial was conducted in an area of very high vector control, good surveillance, and improved access to case management, which we argue are prerequisite to implementing any MDA strategy in similar settings. In lower-transmission areas, infection prevalence among children during the peak transmission season went down to <1%, suggesting that transmission was reduced to a point where elimination may be possible. If these gains can be sustained by continued universal vector control coverage, strong surveillance, and case management, it may be possible to eliminate malaria from this area of Zambia.http://jid.oxfordjournals.org. Consisting of data provided by the author to benefit the reader, the posted materials are not copyedited and are the sole responsibility of the author, so questions or comments should be addressed to the author.Supplementary materials are available at


Plasmodium falciparum (Pf) infections was evaluated. Seventy-two newborns of mothers who were placental malaria negative (PM−) and of mothers who were PM+ with below (PM+ Lo) and above (PM + Hi) median placental parasitemia, were actively monitored during their first year of life. Median time to first PCR-detected Pf infection was shorter in PM + Lo infants (2.8 months) than in both PM− infants  and PM + Hi infants . Total number of new infections was also highest in the PM + Lo group. Only 24% of infants experienced clinical malaria episodes but these episodes occurred earlier in PM + Lo infants than in PM + Hi infants (p = 0.05). The adjusted hazard ratio (95% CI) of having Pf infection was 3.9 (1.8–8.4) and 1.5 (0.7–3.4) for infants in the PM + Lo and PM + Hi groups, respectively. Collectively, low placental parasitemia was associated with increased susceptibility to malaria during infancy. Therefore, malaria in pregnancy preventive regimens, such as sulfadoxine-pyremethamine, that reduce but do not eliminate placental Pf in areas of drug resistance may increase the risk of malaria in infants.Risk of malaria in infants can be influenced by prenatal factors. In this study, the potential for placental parasitemia at delivery in predicting susceptibility of infants to  Plasmodium falciparum (Pf), parasitized erythrocytes adhere to placental villi and accumulate in the intervillous spaces (IVS), causing a condition referred to as placental malaria (PM)1. In addition to eliciting adverse pregnancy outcomes2, PM has been identified as a risk factor for early childhood morbidity and mortality. In general, infants born to PM-positive (PM+) mothers have a shorter time to first Pf infection4 and first clinical episode of malaria6, a higher incidence of Pf infections early in life7, lower hemoglobin levels8, and higher risk of dying9 compared to infants of PM-negative (PM−) mothers. However, the risk of malaria is not homogeneous among infants of PM+ mothers, since some PM+ infants have similar risk outcomes as PM− infants11. There is therefore a need to identify specific prenatal factors associated with increased susceptibility to malaria in infants whose mothers had PM.When pregnant women become infected with the mosquito-borne parasite 3. Likewise, Gabonese infants born to multigravidae were approximately twice as likely to experience clinical episodes of malaria during the first 30 months of life than infants born to primigravidae5. Concerning the timing of infection during pregnancy, when maternal Pf infections occurred within 3 months prior to delivery, Ugandan infants were at a higher risk of infection than when mothers were infected earlier in pregnancy10. It is possible that prenatal exposure to low levels of malarial antigens, rather than higher levels influences early childhood susceptibility since multigravidae tend to have lower parasite loads than primigravidae14 and women who are infected only late in pregnancy would be infected for a short time. No study has directly investigated the influence of placental parasitemia (parasite load in the placenta) on the risk of malaria in infants.Factors thought to be important in modifying the susceptibility of infants to malaria include maternal gravidity and the timing of occurrence of malaria during pregnancy. For example, infants born to multigravid Tanzanian women had their first microscopically detected infection 12 weeks earlier than infants of primigravid womenTherefore, the primary objective of this study was to determine if the susceptibility of Cameroonian infants to Pf was modified by maternal placental parasitemia at delivery. Susceptibility was measured by the median time to first post-natal infection or clinical malaria episode, hazard ratios and total number of times infants were infected during the first year of life. We hypothesized that infants born to mothers with low placental parasitemia (PM + Lo) would have shorter times to first Pf infection and experience more infections during the first year of life than infants whose mothers had higher parasitemias (PM + Hi) or whose mothers were negative for Pf (PM−). Secondarily, to determine if PM + Hi mothers experienced more infections and had higher peripheral parasitemia during the course of pregnancy, thereby potentially exposing their fetuses to higher levels of Pf antigens, maternal peripheral blood samples collected during the course pregnancy were also examined for Pf and umbilical cord blood samples at delivery were tested for Pf parasites and parasite products.Seventy-two infants  that permanently resided in a high malaria-transmission village in Cameroon were followed longitudinally during their first year of life Fig. . At deliP. falciparum infections were detected by microscopy and/or PCR at least once during the first year of life in 89% of infants (64/72). Overall, 55% (35/64) of first infections were detectable by microscopy. The median time to first microscopically detected infection was significantly shorter in infants born to PM+ mothers (4.1 months) compared to infants of PM−mothers (6.4 months), p = 0.009 . Overall, 24% of all infants (17/72) experienced one or more clinical malaria episodes (fever plus blood-smear microscopy positive for Pf) during the first year of life, with 71% of episodes (12/17) occurring after 6 months of age. Because <50% of infants in each follow-up group had clinical malaria, the median time to first clinical episode was not calculated. However, there was a trend for clinical malaria to occur later during the first year of life in the PM + Hi group compared to the PM− group (p = 0.06) and the PM + Lo group (p = 0.05). By 10 months of age, only 5% of PM + Hi infants had experienced at least one clinical malaria episode compared to 22% of PM− infants and 40% of PM + Lo infants. Together, the results suggest that earlier episodes of clinical malaria were likely to occur in PM + Lo infants.Using Cox regression, the effect of placental parasitemia at delivery on the time from birth to Pf infection was controlled for potential confounding factors including gravidity, climatic season when infants were born, hemoglobin genotype of infants, amount of transplacentally acquired anti-Pf merozoite antibodies in cord blood and zone of residence within the village Table . The adjUsing PCR-based genotyping to distinguish between persistent and new infections, a total of 253 new infections were detected in the study cohort. Only new infections were included in the assessment of total number of times infants were infected. When all infants of PM+ mothers, irrespective of parasitemia, were compared with infants of PM− mothers, no significant differences were found  Fig. . HoweverCollectively, the assessment of susceptibility in the infant cohorts demonstrated that low placental parasitemia at delivery was associated with increased susceptibility to malaria during the first year of life.17 were quantified in plasma collected at mid-pregnancy (~5 months)  Fig. . The PM hs) Fig. . A decrein utero. The cord samples were examined for IE by microscopy, Pf-DNA by qPCR and Pf histidine-rich protein-2 (HRP2) by ELISA  of the 80 infants originally enrolled were available to determine if placental parasitemia at delivery was associated with potential for fetal exposure to Pf Studies have described associations between mothers having PM at delivery and an increased risk of their babies having Pf infections. This study focused on the relationship between placental parasite density at delivery and the susceptibility of infants to malaria. The results demonstrated for the first time that infants born to mothers with low, not high, placental parasitemia had a higher risk of malaria infection during the first year of life than infants born to mothers with no placental malaria at delivery. Infants in the PM + Lo group were more susceptible to Pf infection than all infants of PM+ mothers, indicating that much of the PM-associated increase in susceptibility was paradoxically accounted for by low placental parasitemia cases. The association between PM + Lo and susceptibility to Pf remained strong after adjusting for factors related to the environment  and to malaria resistance . Although only a few cases of clinical malaria were diagnosed, presumably because anti-malarial treatment was administered each time asymptomatic microscopic infections were detected, the risk of clinical malaria also tended to be higher in infants of the PM + Lo group compared to the PM + Hi group.14. Furthermore, previous research has shown that high placental parasite load increases the chances of detecting parasites in cord blood18. In the current study, IE were detected in the cord blood of one newborn whose mother was PM + Hi and more newborns in the PM + Hi group had Pf-DNA in cord blood than newborns in the PM + Lo group, indicating higher prenatal Pf exposure in PM + Hi newborns. Interestingly, the presence of HRP2 in cord blood was similar between the PM + Hi and PM + Lo groups, possibly because HRP2 circulates for weeks after parasites and Pf DNA have been cleared19. Overall, mothers with low placental parasitemia at delivery had lower parasite load during pregnancy making it likely that their newborns had been exposed to lower amounts of Pf in utero.PM + Lo mothers had lower overall parasite burden than PM + Hi mothers and were, therefore, less likely to have exposed their fetuses to malarial antigens. That is, women with low placental parasitemia at delivery were infected fewer times and had lower average peripheral blood parasitemia during the course of pregnancy compared to women with high placental parasitemia at delivery Fig. . Also, min utero programs the fetus for increased or reduced susceptibility to malaria is important. Peripheral tolerance to Pf antigens can be induced by T regulatory cells (T-regs) and may contribute to increased susceptibility. Fetal CD4+ T cells are biased towards T-regs since 15–20% of CD4+ T cells in fetal lymphoid organs are T-regs compared to <5% in adults21. Pf antigen exposure can induce the expansion of fetal T-regs which, in turn, suppresses antigen-driven responses by T effector cells24. On the other hand, some studies have shown strong T-helper 1 and T-helper 2 effector responses to Pf antigens in newborns of PM+ mothers, indicating that in utero Pf exposure can result in T cell sensitization25. Interestingly, a study in Kenya demonstrated T cell sensitization to Pf in a subset of newborns and T cell tolerance in other newborns within the same cohort26. The tolerant newborns had an approximately 2-fold increase in risk of malaria during the first three years of life compared to the sensitized newborns. However, the specific factors responsible for the dichotomy in fetal immune responses to Pf antigen remained elusive. Here, the data show that low placental parasitemia, in comparison with high placental parasitemia, is associated with increased risk of post-natal malaria and suggest that the outcome of sensitization or tolerance to Pf in utero may be influenced by the amount of Pf to which the fetus is exposed. The lack of cryopreserved cord blood cells in our cohort precluded any direct investigations of the link between placental parasite load and newborn T-cell sensitization/tolerance. Future studies are needed to identify the immune mechanism(s) induced in utero by fetal exposure to different amounts of antigens.The question of how exposure to Pf 27. Thus, a relatively low Pf antigen concentration in utero might stimulate more T-regs than T effector cells leading to overall suppression of response to Pf. In addition, mouse transgenic T effector cells with receptors to OVA, proliferated very little in the presence of low OVA doses and T-regs, but overcame T-reg suppression when the dose of OVA was increased ~100 fold28. Therefore, high amounts of Pf may be required for human T effectors to escape T-reg suppression in utero.Data from animal studies provide clues on how the amount of antigen might skew human T cell responses towards tolerance or sensitization. For example, the antigen dose required to stimulate T-regs in mice is much lower than that needed for T effector cells to proliferateThe results from the current study demonstrate that placental parasitemia at delivery might be an indicator of fetal Pf exposure and potentially predict the susceptibility of infants to malaria. In this study, infants were dichotomized into two groups, i.e., below and above the median parasitemia which was 25 IE/µL of placental IVS blood. Clearly, additional studies are needed to determine if a universal cut-off parasitemia exists and if that threshold should be based on Pf parasite load in IVS blood smears, impression smears, or histological tissue sections. Nevertheless, infants of women who are Pf-negative by IVS blood smear but positive by histology or impression smears might potentially be at a high risk of malaria during infancy.29 or biannually26. 3) New infections were distinguished from persistent infections by parasite genotyping thereby avoiding an overestimation of the number of infections. 4) Finally, maternal malaria was evaluated not just at delivery but also during pregnancy in a subset of mothers.Even though the sample size of infants was relatively small, the current study replicated results from larger studies that have reported a shorter time to infection in infants born to PM+ compared to PM− women. Moreover, the study is unique in many ways. 1) Infections in infants were detected by PCR, in addition to microscopy, which significantly increased detection sensitivity and provided a better estimate of time to first infection. 2) The active monitoring of Pf infections is also reflected in the short interval between follow-up visits, ~1 month, in contrast to previous studies that monitored infants quarterly31. More effective interventions that eliminate Pf in pregnant women are therefore needed to protect both mothers and their offspring.The translational implications of the findings are significant. If low placental parasitemia increases the risk of infant malaria, then malaria control interventions during pregnancy that do not eliminate Pf parasites from the placenta, but only reduce the Pf density, could increase the risk of malaria in the offspring. This is particularly important because there are reports from parts of Africa of decreasing efficacy of sulfadoxine-pyremethamine in eliminating parasites in pregnant women13. A total of 80 mother-newborn pairs were enrolled and the newborns were subsequently monitored during infancy. Early in the study, 8 mothers voluntarily withdrew their infants at ≤4 months of age, leaving 72 infants who were studied during the first year of life. Mothers of 26 infants had been enrolled at ≤14 weeks of pregnancy and followed monthly until delivery13; whereas, the other mother-baby pairs were enrolled at delivery. None of the mothers received intermittent preventive treatment for malaria in pregnancy (IPTp) since the study was performed before the implementation of IPTp in Cameroon33. The overall design of the current study is summarized in Fig. 34 and the rate of mother-to-child transmission of HIV in Cameroon has been estimated to be ~22.1%35. Thus the probability that a mother was HIV-positive and her baby was infected with HIV was low .The study was conducted in the rural village of Ngali II, Cameroon, between January 2001 and May 2005. In this area, Pf transmission is perennial with two rainy  and two dry  seasons. The estimated entomological inoculation rate was 257 infective bites/person/yearDuring the first year of life, infants were seen at 8 time points corresponding to post-natal ages of 1.5, 3, 4, 5, 6, 8, 10 and 12 months. In addition, infants were monitored at home by four village health workers who arranged for sick children to be examined by the project physician. Therefore, each infant was actively monitored. None of the infants used bed nets and none received anti-malarial chemoprophylaxis within 2 weeks prior to each visit as reported by their parents. At all visits, the infant’s axillary temperature was measured and finger-prick blood collected. A clinical episode of malaria was defined as temperature > 37.5 °C plus blood-smear positive for Pf by microscopy. All infants with microscopic parasitemia regardless of body temperature were treated for malaria according to the Cameroonian government policy for treatment of childhood malaria. The study was approved by the institutional review board of Georgetown University and the National Ethics Committee of the Cameroonian Ministry of Public Health. Informed consent was obtained from the mothers of all infants and the study was carried out in accordance with relevant guidelines and regulations.Blood smears were made from maternal peripheral venipuncture samples collected monthly during pregnancy. The blood smears were stained with Diff-Quick  and examined for IE by microscopy. Pf parasitemia (IE/µL) was determined by counting the number of IE per 200 white blood cells (WBC) and multiplying by the woman’s WBC count.36. Briefly, the umbilical cord was quickly clamped at its placental insertion and a small tissue biopsy was made on the maternal side of the placenta, allowing IVS blood to pool at the excision site. The pooled IVS blood was aspirated into heparinized tubes. Cord blood was drawn directly from the umbilical vein. In previous studies, microsatelite DNA typing of paired IVS and cord blood samples collected using the same method did not show any significant admixture of maternal and fetal blood37. To determine parasitemia in placental IVS blood smears, the number of IE/µl was obtained by counting the number of IE per 200 IVS WBC and multiplying by the woman’s IVS WBC count. Furthermore, placental impression smears and histosections were made from placental biopsies and were semi-quantitatively examined for presence or absence Pf parasites. When IE were detected in histological sections or impression smears, but were not present in IVS blood smears, a parasitemia of < 14 IE/ µl  was assigned to the sample.At birth, about 2 mL placental IVS blood, 5 × 5 × 5 cm placental tissue biopsy and 5 mL of cord blood were collected. The IVS blood was obtained using the placental biopsy-pool method as previously described38, with minor modifications: DNA was extracted from 100 µL of packed red cells premixed with 100 µL of PBS; and, GoTaq master mix  and SsoAdvanced SYBR-Green Supermix  were used for the PCR and qPCR steps, respectively. DNA from in vitro 3D7 parasites was quantified by Nanodrop and serially diluted (10 fold) to make standard curves.The amount of Pf DNA in cord blood was determined by nested quantitative PCR (qPCR), consisting of a conventional PCR step followed by qPCR. The assay was performed as previously reported40. Amplicons were electrophoresed through 1.5% agarose gel (5 V/cm) and SYBR-stained Pf bands were visualized. All Pf DNA-positive samples were evaluated for polymorphisms in the K1, MAD20 and R033 allelic families of Pf MSP1 block 2-4, and in the FC27 and 3D7 allelic families of Pf MSP240. An infant was considered to have a “new infection” if one or more Pf alleles were detected that had not been present at the preceding visit. Finally, standard strip-based electrophoresis was used for hemoglobin (HbAA and HbAS) genotyping using infant peripheral blood samples.During the first year of life, Pf infections were detected by microscopy and by nested PCR using finger-prick peripheral blood samples. DNA was extracted on spin columns using 200 µL of blood and the Pf 18 S ribosomal RNA gene was amplified17.To assess the degree of protection from PM during pregnancy, the levels of IgG and proportion of high avidity IgG antibodies to full-length VAR2CSA at ~5 months of pregnancy were measured as previously describedin utero, cord blood was tested for IgG to Pf MSP142 (FVO strain) and EBA175 antigens by Luminex assay. Briefly, 1 µg of each recombinant antigen was coupled to 1 million beads with a different spectral address per antigen25 and then 50 µL of beads (i.e. 3000 beads for each antigen) was mixed with 50 µL of diluted plasma (1:100 in PBS-1%BSA) and incubated for 1 h on a shaker at room temperature. After two washes with PBS-0.05% Tween and one wash with PBS-1%BSA, 100 µL of 1:250 dilution of 2 µg/ml phycoerythrin-conjugated goat anti-human IgG was added to each well and incubated for 1 h. Beads were washed, resuspended in 100 µL of PBS-1%BSA and analyzed in the LiquiChip 200 analyzer. Results are reported as median fluorescence intensity (MFI).To determine the level of anti-Pf antibodies that was passively acquired by infants To determine if the Pf antigen HRP2 was present in cord blood, samples were screened using the CELISA kit . Whole blood was prepared by recombining equal volumes of cyropreserved plasma and packed red blood cells. The assay was conducted in duplicate using 100ul of blood per well following the manufacturer’s instructions. Serial dilutions of a positive-control sample provided in the kit were used to generate standard curves and the cut-off for positivity were established as specified by the manufacturer.5; residence and season at birth41; HbAA versus HbAS42; and cord plasma IgG to Pf merozoite antigens MSP1 and EBA17544. The cord Pf IgG levels were stratified into 3 categories: > 67th percentile, 33rd to 66th percentile and < 33rd percentile. Ranking was done because unit changes in Pf IgG median fluorescence intensity  were not expected to have significant effects.Kaplan-Meier survival curves and log-rank tests were used to compare the median time to first infection among infant study groups. Right-censoring was done either at withdrawal from study, death, age 1 year, or at end of study period, whichever occurred first. Cox regression was used to assess hazard ratios and 95% CI. Hazard ratios were adjusted for covariates that could potentially influence susceptibility to infection, including gravidityOther data were presented as percentages or as medians and interquartile ranges, unless otherwise stated. Fisher’s exact test was used to compare categorical data. Because many continuous variables were not normally distributed  and owing to the small sample size in some sub-analyses, non-parametric tests were used for group comparisons. The Kruskal-Wallis test was used to compare changes in variables such as average number times infants in the 3 study groups were infected while Mann-Whitney test was used for group-to-group comparisons. Where appropriate, a linear trend test for changes across groups with increasing parasitemia  was applied. P values were expressed to the nearest non-zero number after decimal point and P < 0.05 was considered significant. All P values were two-tailed.The datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Supplementary Figure


Since 2005, Ethiopia has aggressively scaled up malaria prevention and case management. As a result, the number of malaria cases and deaths has significantly declined. In order to track progress towards the elimination of malaria in Amhara Region, coverage of malaria control tools and current malaria transmission need to be documented.Plasmodium falciparum and Plasmodium vivax and serology assays using merozoite antigens from both these species were used to assess the prevalence of malaria infections and exposure to malaria parasites in 16 woredas (districts) in Amhara Region.A cross-sectional household survey oversampling children under 5 years of age was conducted during the dry season in 2013. A bivalent rapid diagnostic test (RDT) detecting both P. falciparum and P. vivax infection was 1.5 and 0.4%, respectively, of which 0.05% presented as co-infections. Overall age-adjusted seroprevalence was 30.0% for P. falciparum, 21.8% for P. vivax, and seroprevalence for any malaria species was 39.4%. The prevalence of RDT-positive infections varied by woreda, ranging from 0.0 to 8.3% and by altitude with rates of 3.2, 0.7, and 0.4% at under 2000, 2000–2500, and >2500 m, respectively. Serological analysis showed heterogeneity in transmission intensity by area and altitude and evidence for a change in the force of infection in the mid-2000s.7878 participants were included, with a mean age of 16.8 years (range 0.5–102.8 years) and 42.0% being children under 5 years of age. The age-adjusted RDT-positivity for Plasmodium vivax infections appear to be lower but relatively more stable across geography and altitude, while P. falciparum is the dominant infection in the higher transmission, low-altitude areas. Age-dependent seroprevalence analyses indicates a drop in transmission occurred in the mid-2000s, coinciding with malaria control scale-up efforts. As malaria parasitaemia rates get very low with elimination efforts, serological evaluation may help track progress to elimination.Current and historic malaria transmission across Amhara Region show substantial variation by age and altitude with some settings showing very low or near-zero transmission. The online version of this article (doi:10.1186/s12936-017-1884-y) contains supplementary material, which is available to authorized users. Plasmodium falciparum and Plasmodium vivax are present and malaria transmission is generally unstable, with focal, seasonal outbreaks and occasional epidemics [Malaria is a major public health challenge in Ethiopia, accounting for approximately 12 million cases each year [During the last decade, significant scale-up of interventions—including population-wide distribution of free, long-lasting insecticide-treated nets (LLINs), indoor residual spraying (IRS), nationwide roll-out of rapid diagnostic tests (RDTs) and artemisinin-based combination treatments—to reduce malaria transmission was implemented throughout the country . This reP. vivax, require a larger specimen volume [P. falciparum and 0–4.5% for P. vivax) [To facilitate malaria elimination in geographical areas with historically low transmission , it is in volume , 13. Ser. vivax) .P. falciparum and/or P. vivax using a population-based survey with malaria RDTs and malaria serology.A collaborative effort between the Federal Ministry of Health and Amhara National Regional State Health Bureau (ANRSHB); the Malaria Control and Elimination Partnership in Africa (MACEPA), a programme at PATH; and The Carter Center (TCC) was established to develop, implement, and evaluate a comprehensive malaria elimination strategy in selected areas of Amhara Region, Ethiopia. Before the implementation of activities, this study was conducted to estimate 2013 baseline rates of: (1) core malaria interventions including LLIN ownership and use, and IRS coverage; and (2) current and cumulative malaria infections due to P. falciparum and P. vivax. Malaria transmission is seasonal, with the major malaria transmission season being between September and December. For the purpose of this study, woredas (districts) of Amhara were divided into four eco-epidemiological zones  based on weekly malaria incidence data from 2012. A total of 16 woredas from these four unique eco-epidemiological zones were selected , PATH, and Emory University.woreda level in 16 target woredas. Woreda-stratified sampling was used to draw a sample from each woreda that was proportional to the woreda population. Within each woreda, enumeration areas (EAs) were selected with probability proportional to size. A total of 278 EAs were selected from woreda listings of all EAs developed by the Central Statistical Agency for the 2007 census. In each selected EA, 25 households were selected using simple random sampling from a sampling frame of all households created by EA mapping. EAs were mapped using personal digital assistants (PDAs). Within each sampled household, all children aged 6 months to 4 years old were eligible for malaria testing. Household members over 5 years of age were eligible in one out of every four households. Individuals were invited to participate and were included after verbal informed consent was obtained.A cross-sectional household survey was conducted in March–April 2013 during the dry, low-transmission season to provide estimates of malaria burden and coverage of malaria control tools at A standardized questionnaire to collect information on socio-demographic characteristics, knowledge and attitudes about malaria, and use of malaria control tools was administered to each participant. Data were collected using PDAs. Finger-prick blood samples were collected from consenting participants to perform an RDT  and prepare dried blood spots (DBS) on filter paper. Participants with a positive RDT result received antimalarial treatment per national guidelines. Those seriously ill were referred to seek immediate care at the nearest health facility.P. falciparum and P. vivax merozoite surface protein (MSP-119) and apical membrane antigen 1 (AMA-1) recombinant protein [Samples were assayed for anti-malarial IgG antibody responses to tein MSP-9 and api protein , 17. PlaData analysis was done using Stata . Estimates of seroprevalence and prevalence of malaria infection by RDT were weighted to account for the sampling design.Plasmodium falciparum and P. vivax seropositivity was defined as an individual being positive for either or both respective antigens, and was analysed by age, altitude, and woreda. Simple reverse catalytic models were fitted to seroprevalence and age data using maximum likelihood methods and seroconversion rates (SCR) were estimated [Serology samples with duplicate OD values that varied by more than 50% were excluded from the analysis. Duplicate OD results were averaged, the raw ODs were transformed into titres using the standard curve generated by the positive control on each assay plate. Seropositivity was calculated by fitting a Gaussian distribution to normalized OD values using maximum-likelihood methods assuming two distributions, a narrow seronegative, and broad seropositive . For eacstimated . Evidencstimated .P. vivax and/or P. falciparum  as compared to having a negative RDT test result, for sex, age, altitude, fever, malaria history, bed net ownership, bed net use, and IRS spraying in the last 12 months. Univariate and multivariate models including all variables to estimate adjusted ORs were run. Two independent models were run for altitude (<2000 and ≥2000 m) because there was a significant effect modification between altitude and bed net use/ownership and altitude and IRS. Moreover, the national malaria programme uses the 2000 m altitude cut-off for targeted deployment of malaria control tools, hence supporting the a priori plan to run the two models separately. Also, it was not possible to add a third altitude band above 2500 m for the regression as was done for the descriptive analysis because the number of RDT positives was too small. Similar regression models were employed for P. falciparum and P. vivax seropositivity.Logistic regression was employed to determine the likelihood of being RDT-positive for woredas  for P. vivax, and 4 (0.05%) for both P. falciparum and P. vivax. Among RDT-positive individuals, 41.0% were asymptomatic (no reported fever in the last 2 weeks), with no significant differences by age group. The overall seroprevalence was 39.4% (n = 3102), with 30.0% (2367) being P. falciparum seropositive and 21.8% (1720) P. vivax seropositive.A total of 7878 individuals from 16 das Fig. ; 120   , 0.3 and 0.4% (2000–2500 m), and 0 and 0.4% (≥2500 m) , 19.8 and 18.9% (2000–2500 m), and 10.7 and 10.6% (≥2500 m) . The age seroconversion plots for antibody responses for P. falciparum and P. vivax by altitude strata are shown in Additional file The age seroconversion plots for antibody responses for The prevalence of RDT-positivity, reported fever and seroprevalence decreased with increasing altitude . However, all seropositive cases seen above 2500 m are borderline positive, as they are near the limit of detection of 0.02 OD units.Additional file Plasmodium infection in nearby low-land farming areas of Amhara [The overall RDT-positivity in the study area during the dry season (March–April) was 1.9%, when the prevalence of infection is expected to be significantly lower than during the major malaria transmission season, which is between September and December in Amhara region. This low prevalence of parasitaemia is probably an underestimate due to the imperfect RDT sensitivity, however it confirms that ongoing transmission persists in this region at a low level into the dry/low transmission season. Another study conducted in July 2013 at the end of the dry season found a high prevalence (12.0%) of RDT-diagnosed f Amhara . The usef Amhara .RDT-positivity was highest among children 0–9 years of age, although this pattern was only seen in the lowest altitude areas. The higher prevalence of infection in this young age group is likely to be driven by lower levels of naturally acquired immunity, with higher parasitaemia densities and potentially longer persistence of parasitaemia at a level that could be detected by RDTs . This shP. falciparum and P. vivax antigens, reflecting the cumulative exposure to these infections over time, given that, once acquired, antibody responses to MSP-119 and AMA-1 can persist for several years [Seroprevalence reflects cumulative exposure to malaria, and, with the antigenic targets used in this study, is expected to fluctuate less between the different transmission seasons. As expected, seroprevalence increased with age for both al years . Thus, sP. falciparum and P. vivax decreased with increasing altitude, which reflects the well-known phenomenon that transmission is generally higher at lower altitudes. In areas below 2000 m, RDT positivity for P. falciparum was higher than for P. vivax . However, at altitudes over 2500 m the opposite was found: the RDT prevalence for P. vivax was 0.4 and 0% for P. falciparum, which may be related to the fact that P. vivax can have persistent liver stages and infection relapses and is also more tolerant of lower ambient temperatures [P. vivax infections is higher than for P. falciparum particularly in low transmission intensity areas [The RDT-positivity and the seroprevalence for both ty areas .P. falciparum was not detected by microscopy and P. falciparum positivity by RDT was of 0.3%, showing the variation due to the different sensitivities of the tests used and highlighting the challenges of monitoring transmission in very low transmission areas.The 2011 Malaria Indicator Survey (MIS) found the same altitude pattern, with a 13-fold higher prevalence of malaria infection in areas under 2000 m, as compared to higher altitudes [P. falciparum RDT-positivity and seroprevalence in areas under 2000 m suggests that these areas are driving the transmission in the Amhara Region by exporting infections to the higher altitude areas. At elevations over 2000 m, RDT prevalence was almost zero, and P. falciparum and P. vivax seroprevalences, which were nearly equivalent, were much lower. All age groups showed seropositivity, although seropositive children in areas above 2500 m had very low antibody titers. This suggests that local transmission in higher altitude areas is very low and that exposure to infection is likely to be acquired elsewhere and imported as a result of the population’s mobility and/or migrant labour workforce [The dominance of orkforce , 28.Finally, seroconversion rates showed a statistically significant change in the force of malaria infection, suggesting that malaria transmission intensity was higher and was reduced nearly tenfold  in the early-to-mid-2000s in Amhara. This finding probably reflects the success of the recent aggressive scale-up of malaria control activities in the area .In a low transmission setting such as Amhara, the typical malaria age pattern, where younger individuals, especially children, have the highest risk of infections, is also observed. Although reported fever in the past 2 weeks was a significant predictor of RDT-positivity, 63.1 and 46.4% of RDT-positive individuals in altitudes <2000 and ≥2000 m, respectively, did not report a history of fever. This suggests that naturally acquired immunity is attained early in life . SimilarHousehold bed net ownership was significantly protective against both RDT positivity and seropositivity at altitudes under 2000 m, but not at 2000 m and above, primarily because bed net distribution programmes were focused specifically in lower altitudes where malaria transmission is known to be higher. Above 2000 m, IRS in the past 12 months was protective against RDT positivity, but was a significant risk factor for seropositivity. This may be due to the fact that certain IRS targeting programmes were implemented in areas ≥2000 m based on previous malaria outbreaks, and therefore may have masked the true effect of IRS spraying.P. falciparum compared to P. vivax and substantially higher transmission in lower altitude areas. Progress made in Ethiopia during the last decade in decreasing malaria transmission should continue with effective interventions tailored to the socio-epidemiological characteristics of the area. Given the apparent importance of population mobility in the parasite transmission patterns, specific interventions targeting the source areas at lower elevations and the mobile population would be warranted to eliminate transmission in the highest altitude areas. Serology offers an opportunity to monitor transmission in low and seasonal transmission areas. Measuring changes in the transmission of malaria will be crucial to monitor progress toward malaria elimination, and serological surveys may be a critical tool in addition to infection detection to describe changes in malaria transmission.As observed in other malarious areas –34, therAdditional file 1. Age-seroconversion plots for antibody responses to Plasmodium falciparum antigens (left) and P. vivax antigens (right) for altitudes below 2000 metres (A), 2000 to 2500 metres (B), and greater than 2500 metres (C). Triangles represent deciles of observed data, solid lines represent the fit to the data of a reverse catalytic conversion model and broken blue lines provide the 95% confidence interval for this fit.Additional file 2. Percent of RDT-positivity , serology positivity and presence of fever by bed net ownership, bed net use and IRS use, in different altitude strata.Additional file 3. Percent and odds ratios for RDT-positivity  by sociodemographic characteristics and malaria risk factors, for altitudes <2000 metres and ≥2000 metres.Additional file 4. Percent and odds ratios for seropositivity  by sociodemographic characteristics and malaria risk factors, for altitudes <2000 metres and ≥2000 metres.Additional file 5. Dot plots of seropositivity to Plasmodium falciparum antigens among children aged 1 to 5 years, by altitude.


Plasmodium falciparum and Plasmodium vivax malaria pathogenesis, fewer have been conducted in India.Malaria remains an important cause of morbidity and mortality in India. Though many comprehensive studies have been carried out in Africa and Southeast Asia to characterize and examine determinants of P. falciparum and P. vivax infection on univariate analysis.A prospective study of malaria-positive individuals was conducted at Goa Medical College and Hospital (GMC) from 2012 to 2015 to identify demographic, diagnostic and clinical indicators associated with P. vivax and P. falciparum cases present year-round. Some 1116 malaria-positive individuals , 88.2% of whom were born outside of Goa and 51% of whom were construction workers, were enroled in the study. Of 1088 confirmed malaria-positive patients, 77.0% had P. vivax, 21.0% had P. falciparum and 2.0% had mixed malaria. Patients over 40 years of age and with P. falciparum infection were significantly (p < 0.001) more likely to be hospitalised than younger and P. vivax patients, respectively. While approximately equal percentages of hospitalised P. falciparum (76.6%) and P. vivax (78.9%) cases presented with at least one WHO severity indicator, a greater percentage of P. falciparum inpatients presented with at least two  and at least three  severity features. There were six deaths among the 182 hospitalised malaria positive patients, all of whom had P. falciparum.Between 2012 and 2015, 74,571 febrile individuals, 6287 (8.4%) of whom were malaria positive, presented to GMC. The total number of malaria cases at GMC increased more than two-fold over four years, with both P. falciparum.During the four year study period at GMC, the number of malaria cases increased substantially and the greatest burden of severe disease was contributed by  Plasmodium genus infect more than 200 million people and cause an estimated 438,000 deaths annually , 43P. fap < 0.01), lower Glasgow coma score, higher blood urea nitrogen, higher serum creatinine, renal failure, per cent parasitaemia, severe anaemia, more days in the hospital and hospitalisation for more than eight days were all significantly correlated (p < 0.05) with P. falciparum infection. Shock or circulatory collapse was the only feature that showed a greater frequency among hospitalised P. vivax cases as compared to P. falciparum, albeit numbers were low in both species of infections. Though hypoglyemia and acidosis were not routinely measured, one case of hypoglycaemia was observed in a P. falciparum patient , while it was not seen among any of the tested P. vivax patients . Metabolic acidosis, when tested for, was observed more frequently in P. falciparum cases  as compared to P. vivax .In addition to increasing clinical severity  reported 4854 cases of malaria in the entire state of Goa, 443 of which were caused by P. falciparum, and showed a more than two-fold decline in cases between 2012 (1714 cases) and 2015 (786 cases) [Between 2012 and 2015, GMC saw 6277 malaria-positive cases, with the number of P. vivax and P. falciparum patients were seen year-round at the GMC. While the year-round presence of P. vivax cases may be explained by the ability of P. vivax parasites to lie dormant as hypnozoites in liver stages with small numbers re-emerging during the dry season, this would not be the case for P. falciparum [P. vivax and P. falciparum may be aided by the recent discovery of a previously unsuspected vector in this region, Anopheles subpictus [Anopheles stephensi peaks, but also in the dry season when An. stephensi numbers are low.Despite the expected increase in number of malaria cases during and after the monsoon season, both lciparum , 45. Altubpictus . This veP. vivax infections were much more common at GMC, P. falciparum infections were associated with greater severity. Although approximately equal proportions of hospitalised patients with P. falciparum and P. vivax had at least one WHO severe malaria feature, patients with two or more severity criteria were more common to P. falciparum cases. The six deaths that occurred among study participants were all P. falciparum patients. In recent years, there have been a number of studies pointing to the importance of severe P. vivax in Asia, with some suggesting that P. vivax may be as or nearly as virulent as P. falciparum [P. vivax caused some severe illness, multi-organ involvement was not typical. P. falciparum infections showed greater severity with substantial multi-organ involvement.Although lciparum –52. In tP. falciparum and P. vivax malaria than adult females. The same gender bias was smaller for children enroled in the study, with 64.7% male children and 35.3% female children. Such findings are consistent with previous reports of age-dependent sex-bias in clinical malarial disease in hypo-endemic regions [There was a strikingly greater proportion of adult males (86.7%) presenting to GMC with  regions  and withP. falciparum and P. vivax patients over 40 years of age were significantly more likely to be hospitalised by GMC than those between the ages of one and 20 years and 21 and 40 years, which points to a possible increase in the susceptibility of older populations to severe malaria in lower transmission settings. Future analysis will explore age-related symptomology and outcomes as well as parasite and gametocyte carriage to better understand if malaria virulence is increased in older populations in Goa. Other studies have shown that presenting syndromes in severe malaria depend on age and that age is an independent risk factor for a fatal outcome of malaria [Both  malaria , 54, 55.P. falciparum, in low prevalence, relatively prosperous areas such as Goa [P. vivax as P. falciparum. These findings may have implications for the conventional understanding of risk factors as well as the basis of targeted control measures at and around construction sites in the low transmission state of Goa. In the future, investigation to determine the possible effect of pre-existing P. falciparum and P. vivax immunity, compared across age, gender, origin and occupation, on transmission in Goa will be carried out.Construction sites in India are commonly suggested as potential transmission hot-spots, especially of severe h as Goa , 56. ThoP. vivax disease, as compared to severe P. falciparum disease, globally or on the Indian subcontinent [P. falciparum infection was used for both species, as is generally the case, which may bias or weaken associations seen among P. vivax cases. The present study does not include data for splenomegaly, spleen rupture or thrombocytopaenia, which have been reported to be common among severe P. vivax cases and are not reflected in the WHO criteria [P. vivax, were not included in the present study. This may impact the disease burden being reported among P. vivax and/or P. falciparum cases. It was also not always possible to accurately determine whether a patient had co-infections and/or had been treated with anti-malarials prior to presentation at GMC, a tertiary care centre. Co-infections and/or prior treatment may have impacted the measurement of clinical severity and/or course of malaria infection [There are a number of limitations to the present study. Due to logistical limitations and the high number of cases, only 18% of the hospital-identified malaria positive cases that presented to GMC over the study period were enroled. To date, there is not a complete, widely agreed-upon definition of what clinical features constitute severe ontinent , 57. As criteria , 24, 52.criteria , 47, whinfection . Future P. falciparum and P. vivax patients at the only government tertiary care facility in a peri-urban setting in southwestern India. The number of malaria cases presenting to GMC increased more than two-fold from 2012 to 2015, which may be of interest in the context of elimination efforts. The data from GMC show a greater burden of disease contributed by P. falciparum than P. vivax and suggest a potential age-dependent increase in susceptibility that will be further investigated.The present study highlights the demographic, diagnostic and clinical profiles of


We characterized posttravel hospitalizations of citizens returning to Israel by summarizing the returning traveler hospitalization dataset of the national referral Center for Travel Medicine and Tropical Diseases at Sheba Medical Center in Israel. Of 722 hospitalizations, 181 (25%) infections were life-threatening; most would have been preventable by chemoprophylaxis and pretravel vaccination.  International travel, particularly to tropical regions and low-income countries, may be associated with the risk for acute illness and hospitalization , patients were included regardless of the time interval since return. We defined nonspecified febrile illness as a febrile illness with an undetermined cause , dengue (74 [10%]) and enteric fever (59 [8%]). Among 145 hospitalized malaria patients, 86 (59%) tested positive for P. falciparum malaria (n = 86) were older than those positive for P. vivax (n = 36)  and were more likely to be business travelers (39 [45%] vs. 1 [3%]); p<0.01); male ; and to have traveled to middle and western Africa . The annual number of P. vivax malaria hospitalizations declined during the study period from an average of 7.3 hospitalizations per year during 2004–2006 to <1 hospitalization per year during 2013–2015 (R2 = 0.62).Patients hospitalized for P. falciparum malaria (86 [48%]) and enteric fever (59 [33%]). Of the 74 cases of dengue fever, none were dengue hemorrhagic fever or dengue shock syndrome; therefore, no dengue cases were considered life-threatening. Eleven (2%) hospitalized travelers required admission to an intensive care unit, and 2 of these patients died during their hospitalization. One patient died from endocarditis caused by Staphylococcus aureus and 1 from necrotizing fasciitis in a surgical wound while hospitalized for eosinophilia and abdominal mass.Of the 181 acute life-threatening tropical diseases, 86 (48%) were acquired in Africa and 83 (46%) in Asia . Male seP. falciparum malaria ; Salmonella enterica serovar Typhi ; hepatitis A ; and acute hepatitis B . Most of the life-threatening diseases acquired in Africa were potentially preventable ; significantly fewer  from Asia were potentially preventable (p<0.01).A total of 112 (16%) hospitalizations were potentially preventable by chemoprophylaxis or pretravel vaccination: We reviewed >700 posttravel hospitalizations of citizens in Israel during 2004–2015. Acute, life-threatening illnesses necessitated 25% of admissions, most of which were potentially preventable by malaria chemoprophylaxis or pretravel vaccination. Compared with other regions, nearly all life-threatening diseases in travelers returning from Africa were preventable.P. falciparum malaria. The number of P. vivax malaria hospitalizations declined during the study years, possibly related to discontinuation of rafting trips to the Omo River in Ethiopia, which had resulted a high number of infections in earlier years. In travelers returning from Asia, enteric fever was the second most common cause of hospitalization, after dengue fever; S. enterica ser. Paratyphi, for which an effective vaccine is not available, caused most of those illnesses. An outbreak of Salmonella Paratyphi A enteric fever in Nepal (Most travelers who were admitted to hospitals to treat preventable life-threatening diseases after returning from Africa were diagnosed with Our study has several limitations. SMC is the national referral center for travel-related illness; therefore, an unusually high number of severe or complicated illnesses may have affected our results. Because of the relatively small number of hospitalizations related to individual destination countries, singular events or large outbreaks may have biased the country-specific data (In conclusion, Israeli citizens hospitalized to treat life-threatening diseases after returning from travel to Africa were likely to suffer from preventable illnesses. Knowledge of region-specific hospitalization causes and impact should be used to identify at-risk travelers, enhance pretravel preparation, and advocate adherence to recommended vaccines and malaria prophylaxis.Detailed information on travel-associated hospitalizations by region of travel  Table 1