Malaria transmission in Latin America is typically characterized as hypo-endemic and unstable with ~170 million inhabitants at risk of malaria infection. Although Colombia has witnessed an important decrease in malaria transmission, the disease remains a public health problem with an estimated ~10 million people currently living in areas with malaria risk and ~61,000 cases reported in 2012. This study aimed to establish the malaria prevalence in three endemic regions of Colombia to aid in designing new interventions for malaria elimination.A cross-sectional survey was conducted in three regions of Colombia with different malaria epidemiological profiles: Tierralta (Ta), Tumaco (Tu) and Buenaventura (Bv). The Annual Parasite Index (API) was 10.7, 6.9 and 3.1, respectively. Participants were asked to respond to a sociodemographic questionnaire and then were bled to determine the Duffy genotype and the prevalence of malaria infection by microscopy and quantitative real-time PCR (qPCR).P. vivax and 22% for P. falciparum, and 4% were mixed infections, which correlates to the overall parasite prevalence in Colombia. This study showed that in the southern Pacific coast of Colombia (Bv and Tu), around 56% of the population have a Duffy-negative genotype, compared to the northern region (Ta) where the percentage of Duffy-negative genotype is around 3%.The study was conducted between October 2011 and January 2012. Eight sentinel sites with 1,169 subjects from 267 households were included. The overall prevalence of sub-microscopic infections measured by thick blood smear (TBS) was 0.3% (n = 4) whereas by qPCR it was 9.7% (n = 113), with a greater proportion (13%) in 40-50 years old individuals. Furthermore, different regions displayed different prevalence of sub-microscopic infections: Bv 12%, Ta 15%, and Tu 4%. From these 113 samples (qPCR), 74% were positive for Sub-microscopic infections are prevalent across different regions in Colombia, particularly in areas with relatively low transmission intensity. The poor microscopy results suggest the need for more sensitive diagnostic tools for detection of sub-microscopic infections. This study underscores the importance of conducting active case surveillance to more accurately determine malaria incidence, and highlights the need for updating the malaria guidelines to track and treat sub-microscopic malaria infections. The burden of malaria has declined in recent years across a number of countries as a result of increasing funding and political commitment, implementation of new anti-malarial treatments, better access to diagnostics, and the extended use of insecticide-treated bed nets and indoor residual spraying -3. This Plasmodium infection varies from asymptomatic to severe and fatal malaria. Continuous exposure to Plasmodium parasites leads to clinical immunity and asymptomatic carriers [Clinical manifestation of carriers ,5. In adcarriers -9.Although microscopic diagnosis continues to be the ‘gold standard’ technique for malaria, including certification of malaria elimination , moleculAlthough asymptomatic infections have not been extensively studied in Colombia, there is growing evidence that sub-clinical infections are more common in the region than previously thought ,17. ThisThree areas were selected in Colombia. Sentinel sites in each of these areas were selected based on the annual parasite index (API), accessibility and safety.Plasmodium vivax (82.4%) followed by Plasmodium falciparum (17.4%) and mixed malaria infections (0.2%) [Tierralta is a municipality located in the Department of Cordoba in the northwestern part of Colombia with ~90,000 inhabitants composed of ~2% indigenous groups and 44.4% rural population. The Annual Parasite Index (API) in 2011 was 10.7. The predominant malaria parasite species in this region is s (0.2%) . The sel~187,084 inhabitants composed mainly of Afro-descendants (88%). The API in 2011 was 6.9. The predominant malaria parasite species in the region is P. falciparum (79.2%) followed by P. vivax (20.8%) [. The sentinel sites selected in this region were Robles, Candelillas and Bucheli.Tumaco is located in the Department of Nariño in the southwest of the country near the Ecuador border. It has a population of P. vivax (85%) followed by P. falciparum (15%) [Buenaventura is located on the Pacific coast of the Valle Department in the west of Colombia, with ~350,000 inhabitants who are predominantly Afro-descendants (72.4%). The API in 2011 was 3.1. The predominant malaria parasite species in the region is um (15%) . The senThe study was a cross-sectional survey in three malaria-endemic regions of Colombia, which is one of the countries in the network of the Latin American Center for Research on Malaria (CLAIM) established with the National Institutes of Health (NIH) funding in 2010. The study was conducted between October 2011 and January 2012 in eight sentinel sites.The study was developed in two steps: first, a population census was performed with door-to-door visits and the help of community leaders. The number of houses, people/house, age, and sex were recorded. Based on this information, a probabilistic sample was calculated for each sentinel site with a confidence level of 95%, and error of 2% and an estimated prevalence of 2%. Second, a set of houses was randomly selected, and every person from the household present at the moment of sampling answered a questionnaire regarding symptoms and epidemiologically relevant information. Males and females of all ages were included. No others visits were made.Blood samples were collected by finger prick on filter paper for malaria parasite DNA isolation and qPCR diagnosis. For Duffy genotype determination, a blood sample of 500 μL was collected by venipuncture from every subject and stored in EDTA containing tubes. Samples were handled as potential biohazards and all laboratory staff strictly followed bio-safety standardized procedures.Approximately 100 μL of blood were collected by finger prick and both thick and thin smears were prepared and stained by Giemsa method for malaria diagnosis. Each slide was examined separately by two experienced microscopists, recording species and densities of sexual and asexual forms. In positive films, parasite species were identified and density recorded as the number of parasites per 200 white blood cells (WBC). Two-hundred high-power fields of the thick films examined at 1,000× magnification were read before recording a negative result. Inter-observer discordances were solved with the reading of a third microscopist. Subjects were considered positive for malaria only if two of the three readings were positive.P. falciparum and P. vivax DNA positive and negative controls were used in each batch of tests, including the extraction of both negative and inhibition control. A sample was considered negative if there was no increase in the fluorescent signal after a minimum of 40 cycles. Parasitaemia quantification was performed using a parasite-specific standard curve obtained by diluting P. vivax and P. falciparum field isolates to densities of 20000, 2000, 200, 20 and 2 p/μL, spotted on filter papers and air dried. Each reaction plate included a standard curve for parasite quantification. Positive samples were confirmed by an independent DNA extraction and a new round of qPCR by triplicates. Samples were considered positive with at least two positive wells in the confirmation reaction.Assays were performed as described previously ,20 with et al. [ES, and FY*BES/FY*BES.The sample size was calculated with a confidence level of 95%, error of 5% and an estimated prevalence of the Duffy negative genotype of 30% with a population census of 5,603 for all the sentinel sites. DNA extraction was performed from 200 μL of total blood samples collected in EDTA tubes, using the PureLink Genomic DNA Mini Kit  following manufacturer’s instructions. The Duffy allele genotyping was performed by qPCR using SYBR Green chemistry as described by Sousa et al.  previouset al. . In eachStudy data were collected and managed using REDCap electronic data capture tools hosted at Caucaseco Scientific Research Center . REDCap The data quality assurance (QA) procedure consisted of setting a quality control (QC) sample of size q less than the total number p of all case report forms (CRFs) for each location. The maximum permissible error for this study was set to 1%.Data analysis was performed using MATLAB (version 2011b). Using descriptive statistics, the general characteristics of individuals admitted to the study were established.This study was carried out in accordance with institutional guidelines. The protocol was previously reviewed and approved by the institutional review board (IRB) of Caucaseco Scientific Research Center . Written informed consent (IC) was obtained from each volunteer at enrolment. Parents were asked to consent for their child to take part in the study. Information obtained from the participants was managed on principles of confidentiality. Immediately after blood donation, malaria-positive volunteers were provided with the anti-malarial treatment recommended by the Colombian Ministry of Health.Between October 2011 and January 2012 a total of 1,169 subjects  were enrolled in three malaria-endemic sites. A total of 272 volunteers were recruited in Tierralta, 460 in Tumaco and 437 in Buenaventura  columns; TBS shows the number of positive samples with parasitaemia detected by microscopy (thick blood smear); Samples positive for the presence of P. vivax by qPCR are shown in the indicated column; FY(+) indicates the % of people presenting Duffy positive genotype.The overall malaria prevalence assessed in the three study sites by thick blood smear (TBS) was 0.3% 4/1,169). The prevalences in Tierralta and Tumaco were 0.4 1/272) and 0.7% (3/460), respectively, whereas in Buenaventura no positives slides were detected  in Colombia and FY*B/FY*B the least frequent (6.6%), based on the study population. However Duffy genotype distribution varied considerably among the three malaria-endemic regions. In Tierralta the most common genotype was FY*A/FY*B with 29.3%, followed by FY*A/FY*A (24.2%), FY*B/FY*BES (18.2%), FY*B/FY*B (15.2%), FY*A/FY*BES (10.1%), and finally FY*BES/FY*BES with 3%. Contrasting results were found in Tumaco and Buenaventura, which are located on the Pacific coast, with a higher percentage of black African people than the Atlantic region. In Tumaco the most common genotype was FY*BES/FY*BES with 53.6%, followed by FY*A/FY*BES (21.6%), FY*B/FY*B*ES (13.4%), FY*A/FY*B (5.2%), FY*A/FY*A (4.1%), and finally FY*B/FY*B (2.1%). A similar pattern was found en Buenaventura, being the most common FY*BES/FY*BES with 56.6%, followed by FY*A/FY*BES (14.2%), FY*B/FY*BES (10.4%), FY*A/FY*A (8.5%), FY*A/FY*B (7.5%), and the least common FY*B/FY*B with 2.8%. Table Of the 302 analysed samples, P. vivax is the predominant species. The prevalence of malaria sub-microscopic infections in low-transmission areas could be dependent on the detection method used. This finding may explain the maintenance or persistence of residual transmission foci due to the existence of asymptomatic human reservoirs with parasitaemia that are not regularly detected. Undetected asymptomatic infections do not receive proper treatment, contributing to maintaining the transmission cycle to Anopheles vectors and therefore malaria transmission. Additionally, our results demonstrate the effectiveness of qPCR to detect these parasitaemias. The availability of an efficient and effective technique for detecting these infections would be a key factor for decision-making. Two other studies in Colombia showed the presence of asymptomatic malaria [This study shows that in low transmissions settings of Colombia there is a considerable amount of circulating parasites at low parasitaemias which are not detected by the current methods employed by the malaria control programme. The prevalence of sub-microscopic infections varies largely between 3 to 20%, with a higher rate in regions where  malaria ,17, howeAccurate parasite prevalence determination is key for monitoring malaria interventions. In this study, we provide first evidence showing submicroscopic parasitaemia levels among apparently healthy volunteers in endemic regions of Colombia. Several studies have reported detection of infection by molecular methods. A study in Rondônia, Brazil, showed that PCR was 6–7 times more efficient than microscopy in detecting malaria infections . A previP. vivax, and red blood cells that lack these antigens are resistant to invasion by this parasite but can still be invaded by P. falciparum [P. vivax infections. On the southern Pacific coast of Colombia (Buenaventura and Tumaco), the prevalence of the Duffy-negative genotype was 56%, compared to the northern region  where the corresponding percentage was around 3%. On the other hand, the frequency of Duffy-positive genotype was higher in Tierralta than in Tumaco and Buenventura, these two areas being endemic for P. falciparum and Tierralta for P. vivax. The parasite species distribution correlates with the Duffy genotype prevalence, as P. vivax infection detected among the Duffy positive individuals was five times higher than P. falciparum. The distribution of the negative Duffy genotype on the Pacific coast is consistent with the predominant Afro-descendants and the Mestizo population migration patterns in Colombia. Furthermore, the very low frequency of Duffy negative factor found in the North coast population could be due to a higher percentage of interbreeding with indigenous populations and a low migration pattern of Afro-descendants.The Duffy glycoprotein antigen on red blood cells is a receptor that binds the malaria parasite lciparum . In malaBecause malaria prevalence officially reported by Pan American Health Organization/World Health Organization (PAHO/WHO) is based on official country data, it is significantly underestimated in the region ,26,27. SWhereas there is general agreement about sub-reporting and other critical logistic aspects related to delivering health services, the notion of sub-microscopic malaria that results in undetectable transmission is not recognized as of local or regional importance. Thus, biological components of transmission need to be considered as a crucial part of any research malaria agenda that aims to support elimination. Additionally, targeting sub-microscopic carriers will be a challenge for malaria control programmes. Possible approaches to include sub-microscopic carriers in treatment campaigns include mass screening and treatment of all parasite carriers (MSAT) and mass treatment without prior screening for parasitaemia, i.e., mass drug administration (MDA). MSAT depends on the availability of very sensitive tools that could be used under field conditions. A very low parasitaemia can be detected by molecular methods such as qPCR; however, these techniques require considerable training and are restricted to reference laboratories. Diagnostic tools, such as the Loop mediated isothermal amplification (LAMP), which are capable of detecting very low parasite densities (one parasite/μL) and are reliable under field conditions could be important for active case detection to accelerate malaria elimination . LAMP haMicroscopy continues to be the gold standard method both for malaria routine diagnosis and elimination , howeverThe presence of sub-microscopic malaria infections in different regions in Colombia shown here, especially in areas of low transmission intensity, underscores the importance of conducting active case surveillance to more accurately determine malaria prevalence in endemic regions, as well as to eliminate malaria reservoirs. There is a need to use techniques more sensitive than microscopy-based TBS for detection of these sub-microscopic infections.


Nowadays, the gold standard method for malaria diagnosis is a staining of thick and thin blood film examined by expert laboratorists. It requires well-trained laboratorists, which is a time consuming task, and is un-automated protocol. For this study, Maladiag Software was developed to predict malaria infection in suspected malaria patients. The demographic data of patients, examination for malaria parasites, and complete blood count (CBC) profiles were analyzed. Binary logistic regression was used to create the equation for the malaria diagnosis. The diagnostic parameters of the equation were tested on 4,985 samples . The equation indicated 81.2% sensitivity and 80.3% specificity for predicting infection of malaria. The positive likelihood and negative likelihood ratio were 4.12 (95% CI = 4.01–4.23) and 0.23 (95% CI = 0.22–0.25), respectively. This parameter also had odds ratios . The equation can predict malaria infection after adjust for age, gender, nationality, monocyte (%), platelet count, neutrophil (%), lymphocyte (%), and the RBC count of patients. The diagnostic accuracy was 0.877  (95% CI = 0.871–0.883). The system, when used in combination with other clinical and microscopy methods, might improve malaria diagnoses and enhance prompt treatment. Malaria is a potential medical emergency and should be treated immediately because delays in diagnosis and treatment are leading causes of death in many countries3To gain a more precise diagnosis, malaria is diagnosed using microscopic diagnosis by the gold standard method which is the staining of thin and thick peripheral blood smears8Changes in hematological parameters are well-known features of malaria infection. These changes involve various cell types such as RBCs, leucocytes and thrombocytes1616171820212223st 2008 and December 1st 2012, data from of 43,899 cases of patients suspected with malaria infection was analyzed. Of these cases, 3,082 (7%) were patients with malaria infection, whereas 40,817 (93%) were patients with non-malaria infection according to thick and thin film examinations.Between January 1The regression model was performed to create the equation for malaria diagnosis. In the first, all patients’ parameters previously collected were entered and calculated . Variablz = (−7.499) − 0.373(gender) + 0.018(age) + 1.019 + 0.017(neutrophil) + 0.017(lymphocyte) + 0.022(platelet) − 0.033(monocyte) + 0.691(RBC)(−z)))Prob  = 1/ = 1-Prob Z = constant of regression analysisAfter ROC analysis was performed , the suiIn the first regression step, the parameter that was significant in the equation included gender, age, nationality, neutrophil (%), lymphocyte (%), platelet count, monocyte (%), and RBC. This is due to these parameters incurring significant changes during malaria infection that can result differently from non-infected malaria patients. The previous study of patients in Thailand showed that gender, age, nationality, neutrophil (%), lymphocyte (%), platelet count, monocyte (%), and RBC significantly changed from malaria infections. A previous study showed patients with higher a WBC count compared with community controls6111213An automated blood cell counter (ACC) is a practical tool for malaria diagnosisP. falciparum. It was also lower when compared to the sensitivity and specificity of the Opti-MAL testP. falciparum and specificity of 100% for P. falciparum. Although the efficiency of this software was lower in terms of sensitivity and specificity than those methods currently used in routine laboratories, it served as another way to help diagnose suspected malaria patients. Further studies are required to improve the sensitivity, specificity, and validate this software. In addition, the software, when used in combination with other clinical and microscopy methods, might help to improve malaria diagnoses in endemic areas.Similar to the RDT, this software will reduce the time of malaria diagnosis, with the result obtainable in 5–20 min2627This study protocol was approved by The Ethical Clearance Committee on Human Rights Related to Research Involving Human Subjects of Walailak University. The informed consent was not obtained from all subjects but the name and Hospital Number (HN) of patients were not revealed.st 2008 and December 1st 2012 were extracted and enter into the SPSS program. Demographic, clinical and laboratory data of all the patients were also collected. Leukocyte, red blood cells, and platelet counts were measured using an automatic cell counter. Other laboratory examinations including RBC, Hb, MCV, MCH, MCHC, RDW, and platelet counts were also collected. The standard procedure used for the diagnosis of malaria is the examination of thick and thin blood smears for malaria parasites by the Wright and Giemsa staining under light microscopy by laboratorists at 1,000 magnification.The data used in this study were collected from the Medical Technology Laboratory Unit, Phobphra Hospital, Tak Province. The methods were carried out in accordance with the approved guidelines. The medical record keeping system in the Department at The Phobphra Hospital is electronic. The data of all patients diagnosed with malaria between January 1The Kolmogorov-Smirnov test was performed to test the normal distribution of the data. Binary logistic regression was performed to find out which parameter was suitable for predicting malaria infections; the logistic regression model was performed using the leukocyte count, RBC count, Hb, MCV, MCH, MCHC, RDW, and platelet counts as covariates and using the status of malaria infection (found or not found) as a dependent variable. Data analysis was performed using SPSS ver. 11.5 . Any parameters that were statistically significant will be put into the second step of regression analysis. Diagnostic accuracy of hematological parameters was measured by a receiver operating characteristic (ROC) and then calculating the sensitivity, specificity, positive/negative predictive values and odds ratios with 95% confidence intervals.How to cite this article: Kotepui, M. et al. A diagnostic tool for malaria based on computer software. Sci. Rep. 5, 16656; doi: 10.1038/srep16656 (2015).


Plasmodium knowlesi is a simian parasite that has been recognized as the fifth species causing human malaria. Naturally-acquired P. knowlesi infection is widespread among human populations in Southeast Asia. The aim of this epidemiological study was to determine the incidence and distribution of malaria parasites, with a particular focus on human P. knowlesi infection in Malaysia.Plasmodium species.A total of 457 microscopically confirmed, malaria-positive blood samples were collected from 22 state and main district hospitals in Malaysia between September 2012 and December 2013. Nested PCR assay targeting the 18S rRNA gene was used to determine the infecting Plasmodium species by using nested PCR assay. Plasmodium knowlesi was identified in 256 (56.5%) samples, followed by 133 (29.4%) cases of Plasmodium vivax, 49 (10.8%) cases of Plasmodium falciparum, two (0.4%) cases of Plasmodium ovale and one (0.2%) case of Plasmodium malariae. Twelve mixed infections were detected, including P. knowlesi/P. vivax (n = 10), P. knowlesi/P. falciparum (n = 1), and P. falciparum/P. vivax (n = 1). Notably, P. knowlesi ) showed the highest proportion in Sabah , Sarawak , Kelantan , Pahang , Johor , and Terengganu . In contrast, the rates of P. knowlesi infection in Selangor and Negeri Sembilan were found to be 16.2% (18/111 cases) and 50.0% (5/10 cases), respectively. Sample of P. knowlesi was not obtained from Kuala Lumpur, Melaka, Perak, Pulau Pinang, and Perlis during the study period, while a microscopically-positive sample from Kedah was negative by PCR.A total of 453 samples were positive for P. knowlesi infection, the findings from this study highlight the widespread distribution of P. knowlesi in many Peninsular Malaysia states.In addition to Sabah and Sarawak, which have been known for high prevalence of  Malaria is one of the most widespread diseases in the world and is endemic throughout tropical and subtropical regions. In 2012, there were an estimated 207 million cases of malaria, causing approximately 627,000 deaths[Plasmodium species are known to infect humans: Plasmodium falciparum, Plasmodium vivax, Plasmodium malariae, Plasmodium ovale, and Plasmodium knowlesi[Plasmodium falciparum causes the most severe disease and tends to predominate in tropical areas, whereas P. vivax is the predominant species outside of Africa.Five  knowlesi. PlasmodP. falciparum , P. vivax , P. malariae , P. knowlesi , P. ovale , mix-infection  and 12 (0.25%) malaria-related deaths in Malaysia[Malaria constitutes an important public health problem in Malaysia, especially in the states of Sabah and Sarawak, in Borneo island as well as the central interior regions of Peninsular Malaysia , which have a predominantly aboriginal populations. In 2012, there were 4725 cases of malaria including Plasmodium knowlesi is a malaria parasite infecting Old World monkeys that is prevalent in Southeast Asia[P. knowlesi infections were initially reported in the Kapit Division of Sarawak [P. knowlesi infections occur in Sarawak and Sabah[Anopheles species suspected to be responsible for P. knowlesi transmission such as Anopheles hackeri, Anopheles balabacensis, Anopheles cracens and Anopheles latens[ast Asia. It has ast Asia. A largealaysia), which wnd Sabah,6, Kalimnd Sabah, Peninsund Sabah,9 Thailand Sabah,11, Singnd Sabah, Philippnd Sabah, Vietnamnd Sabah, and Myand Sabah. These ies latens-18.Plasmodium knowlesi carries high potential of causing severe or fatal diseases[P. malariae via conventional microscopy due to similarities in morphology at certain asexual stages. Indeed, microscopic examination of the asexual stages of Plasmodium on thick and thin blood film is unable to distinguish early trophozoites of P. knowlesi from P. falciparum and late erythrocytic stages from P. malariae[P. knowlesi. Thus, in the present study, molecular approach was employed to investigate the epidemiology of P. knowlesi in Malaysia. PCR assay has been described as the most sensitive and specific technique for malarial detection and is capable of identifying mixed infections using species-specific primers[diseases. However malariae. Neverth primers,20.This study was approved by the Medical Research Ethic Committee (MREC) Ministry of Health Malaysia and Medical Ethics Committee of University Malaya Medical Centre. This study was conducted at 22 state and district government hospitals all over Malaysia: Raja Perempuan Zainab II Hospital, Kota Bharu; Sultanah Nur Zahirah Hospital, Kuala Terengganu, Terengganu; Tengku Ampuan Afzan Hospital, Kuantan, Pahang; Kuala Lipis Hospital, Kuala Lipis, Pahang; Jerantut Hospital Jerantut, Pahang; Sultanah Aminah Hospital, Johor Bahru, Johor; Sultanah Fatimah Specialist Hospital, Muar, Johor; Melaka Hospital, Melaka; Tuanku Jaafar Hospital, Seremban, Negeri Sembilan; Tengku Ampuan Rahimah Hospital, Klang, Selangor; Selayang Hospital, Selangor; Sungai Buloh Hospital, Selangor; Raja Permaisuri Bainun Hospital, Ipoh, Perak; Sultanah Bahiyah Hospital, Alor Setar, Kedah; Sultan Abdul Halim Hospital, Sungai Petani, Kedah; Pulau Pinang Hospital, Pulau Pinang; Tuanku Fauziah Hospital, Kangar, Perlis; Sarawak General Hospital, Kuching, Sarawak; Kapit Hospital, Kapit, Sarawak; Kota Bharu Public Health Laboratory, Kota Bharu, Kelantan and the Department of Vector Borne Disease Control Programme, Kota Kinabalu, Sabah Figure .Four-hundred and fifty-seven microscopically positive malaria blood samples (in EDTA tube), blood spots  and blood films were collected from the above locations. Microscopic examination of Giemsa-stained thick and thin blood films were carried out by microscopists of the respective hospitals who have no knowledge of the PCR results. Positive malaria samples were sent by mail express to the Department of Parasitology, Faculty of Medicine, University of Malaya. No randomization was applied to the sampling. Due to logistical, as well as personal co-operative difficulties, the original intention of complete recruitment of all malaria patients was not achieved. Nevertheless, sampling encompassing nine states of Malaysia was successfully done.Templates DNA for nested PCR assay were prepared. DNA extraction was performed on 100 μl blood collected in EDTA-vacutainer (n = 352) using DNeasy Blood & Tissue Kit . DNA extraction from dried blood spots (n = 81) and blood smears (n = 24) were conducted according to protocols described previously,22 to obPlasmodium small subunit ribosomal RNA (SSU rRNA) gene. The primers used for the nested PCR assay were identical to those previously published[P. knowlesi positive samples were sent to Mytacg Bioscience for DNA sequencing to confirm the species.The species of the malaria parasite in the samples was determined by nested PCR assay. This assay targets the ublished. The reaStatistical analysis was carried out using the SPSS software programme for Windows version 17 . A Pearson’s Chi-square test was used to test the association between each variable and the confidence interval (95% CI) was calculated to explore the association between variables of interest. The level of statistical significance was set at p <0.05 for each test.Plasmodium sp. based on nested PCR assay , 29.4% (n = 133), 10.8% (n = 49), 0.4% (n = 2), 0.2% (n = 1), respectively. Moreover, 12 mixed infections were detected, including P. knowlesi/P. vivax (n = 10), P. knowlesi/P. falciparum (n = 1), and P. falciparum/P. vivax (n = 1).A total of 453 out of 457 samples were positive for y Table . Among tP. knowlesi  showed the high prevalence in Sabah , Sarawak , Kelantan , Pahang , Johor , Terengganu . In contrast, the rates of P. knowlesi infection in Selangor and Negeri Sembilan were 16.2% (18 out of 111 cases) and 50.0% (5 out of 10 cases), respectively. Sample of P. knowlesi was not obtained from Kuala Lumpur, Melaka, Perak, Kedah, Pulau Pinang, and Perlis during the study period, while a microscopically-positive sample from Kedah was negative by PCR  were infected with P. knowlesi only, whereas 11 displayed mixed infections involving P. knowlesi . Many cases of single P. knowlesi infection had been microscopically misdiagnosed as other species, including P. malariae , P. vivax , and P. falciparum . Furthermore, 6 mixed infections involving P. knowlesi  were misdiagnosed microscopically as single P. falciparum (n = 3), single P. vivax (n = 2), or single P. malariae (n = 1) infections and five cases of mixed infection (i.e. P. knowlesi/P. vivax) were misdiagnosed microscopically as single P. knowlesi  were diagnosed as P. knowlesi. Notably there were other cases of single P. knowlesi and P. vivax had been wrongly identified as mixed infection; P. falciparum/P. malariae and P. knowlesi/P. vivax respectively  were negative when tested by nested PCR assay.In addition, the PCR method revealed that six cases of y Table . HoweverPlasmodium knowlesi infections were detected within all age groups of the study populations, with the youngest being six months old and the oldest being 82 years old . The infection was more prevalent among adults  had been microscopically misdiagnosed as P. malariae. PCR findings also demonstrated that P. knowlesi were detected in almost all cases of microscopically identified P. malariae infections. P. knowlesi cases that were microscopically misdiagnosed as P. malariae were not found in Pahang and Kelantan. Meanwhile, only three cases of P. knowlesi infection were misdiagnosed as P. malariae in Sarawak. However, most of the P. knowlesi infections in Sabah were misdiagnosed as P. malariae by microscopic examination. The decrease in microscopy diagnostic discrepancy is due to the increased awareness about P. knowlesi infections among the local laboratory technicians and microscopists. Coupled with the fact that P. malariae infection is not frequently found in Malaysia, the encounter of malaria parasites with morphology similar to that of P. malariae has been reported as “P. knowlesi” infection by default. In addition, clinical data such as the relatively high parasitaemia and low platelet reading serve as useful guides in diagnosing P. knowlesi infection as well.Based on preliminary study, it is indicated that P. knowlesi, which sometimes cannot be accurately diagnosed by microscopic examination. The negative nested PCR results yielded from the four microscopically malaria-positive cases were likely to be caused by DNA degradation of the samples. Coincidently, these four samples were DNA templates extracted from the blood smears, indicating the relatively inferior quality of DNA extracted from blood smears, as pointed out by previous studies[From this study, it is clearly demonstrated that PCR represents a more sensitive and specific method for detecting mixed infections, which tend to be overlooked in the microscopy-based assay, especially when one of the species is present at a lower density. Moreover, PCR allows specific identification of  studies,28. Neve studies, especiaP. knowlesi detection in this study were not the most recently developed primers. Issues regarding non-specific binding of these primers to P. vivax were reported previously[P. knowlesi-positive by the Pmk8 and Pmkr9 primers were indeed P. knowlesi-infected samples.The PCR primers (Pmk8 and Pmk9) used for eviously. NeverthP. knowlesi may occur from human to human due to the discovery of family clustering cases[P. knowlesi cases was found in the present study. The infection was more prevalent among adults (Table P. knowlesi. The highest rate of infection (28.5%) occured in patients in the age group of 21 to 30  display morphological similarities to P. falciparum, while other blood stages are identical to P. malariae[P. knowlesi could be misdiagnosed as P. malariae (majority), P. falciparum, P. vivax, or mixed infections . As P. knowlesi displays the shortest replication cycle of all known human and non-human primate malarias (24 hours), malaria caused by this species is potentially fatal[P. knowlesi as P. malariae may result in delayed treatment[Plasmodium species is fundamental for proper clinical management and control strategies. In addition, any case demonstrating “P. malariae-like parasites” should be treated presumably as “P. knowlesi infection” while waiting for the molecular confirmation diagnosis.Early  malariae,9, resully fatal. TherefoP. knowlesi infection, the present investigation highlights the widespread distribution of human knowlesi malaria in Peninsular Malaysia . In fact, P. knowlesi was found to be the most prevalent Plasmodium parasite infecting the population of Sabah and Sarawak  as well as Peninsular Malaysia. Accurate epidemiological data serve as an important foundation for developing effective control strategies for malaria. The present study has provided critical data that contribute to the better understanding on the distribution of P. knowlesi within the interior division of Sabah and Sarawak and central regions of Peninsular Malaysia.In addition to Sabah and Sarawak, which have high record of Consent was granted by patient/patient’s family for the publication of these case reports.The authors declare that they have no competing interests.RY, JJ, HUG, SM, HMH, NM and MMA collected blood samples and patients’ data. RY, and YLL analyzed and interpreted the data. RY, YLL, RM and MYF arranged the data, conceptualized and prepared the manuscript. All authors read and approved the final manuscript.


Plasmodium falciparum malaria is the most common infection in Yemen. The present study aims to investigate changes in hematological and hepatic function indices of P. falciparum infected individuals. This study included 67 suspected falciparum malarial patients attended in clinics and rural Abs Hospital , Yemen, from October 2013 to April 2014. The diagnosis of malaria was confirmed by thick and thin film with Giemsa staining of malaria parasite. Hematological parameters and serum levels of aspartate transaminase (AST), alanine transaminase (ALT), alkaline phosphatase (ALP), and bilirubin  as test indicators of liver function were studied. Patients with parasitaemia tended to have significantly lower hemoglobin, hematocrit, white blood cell count, lymphocytes, and platelets, compared with healthy normal subjects. Neutrophils levels were significantly higher in cases of falciparum malaria in comparison to healthy normal subjects. Serums AST, ALT, ALP, and bilirubin  in falciparum malaria patients were significantly higher (p < 0.0001) than those of falciparum malaria of free individuals. Hematological and liver dysfunctions measured parameters were seen associated with moderate and severe parasitaemia infection. This study concludes that hematological and hepatic dysfunction parameters could be indicator of malaria in endemic regions. Plasmodium which is transmitted to human beings by infected female anopheles mosquito [ P. falciparum is the predominant species in Yemen where it is responsible for more than 90% of the malaria cases, with only minimal cases caused by Plasmodium vivax [ P. falciparum infested blood may vary with level of malaria endemicity, presence of haemoglobinopathies, nutritional status, demographic factors, and level of malaria immunity [Malaria continues to be a great health problem in some of the most populated areas of the world. Infection is caused by a parasite of genusmosquito . In Yememosquito . P. falcum vivax . Studiesum vivax –6. Changimmunity , 8. The immunity . falciparum malaria and is attributed to hemolysis of both parasitized and nonparasitized erythrocytes and partly due to liver damage [ falciparum malaria infected patients in Abs region of Yemen, which suffers from a highly malaria endemic.Liver involvement in malaria is common in patients of severe malaria and may manifest as jaundice, hepatomegaly, and elevated liver enzymes like aspartate and alanine transaminases . Hyperbir damage . Therefo falciparum malarial parasite by microscopic examination of Giemsa stained thin blood smears. Patient's selection was done by simple random sampling of males and females between October 2013 and April 2014. 30 normal subjects free from malaria were used as control. Patients with hepatitis, smokers, and those who are taking any antimalaria drugs were excluded from this study.A total of 67 patients with malaria parasitaemia attending the clinics and Rural Hospital of Abs , Yemen, were enrolled as the test subjects, who were reported ill with fever (temperature > 37.5°C), headache, vomiting, chills, diarrhea, and other clinical signs. Patients of age 18–48 years were selected who confirmed to be infected with theP. falciparum parasitaemia was determined in various blood smears stained by Giemsa stain. Parasitaemia was calculated based on WHO [d on WHO : low (+)Sample collection and preparation: Venous blood was collected aseptically from the subjects using 5 mL disposable syringes. The blood samples were collected and 4 mL was transferred into plain bottles for the biochemical assays whereas the remaining 1 mL was transferred into EDTA bottles for malaria parasite tests. The blood samples in the plain bottles were allowed to clot and retract after which they were centrifuged at 3000 rpm for 10 min and the serum was transferred into sterilized plain bottles for the biochemical analysis.Whole blood samples were collected in EDTA tube for determination of hematological parameters including hemoglobin (Hb) concentration, tWBC count, neutrophils, lymphocytes, platelets, and packed cell volume (PCV) using automated (SYSMX.KX-21n) hematology analyzer.Serum aspartate transaminase and alanine transaminase (ALT) parameters were determined using Roche/Hitachi cobas c systems automatically. This assay follows the recommendations of the IFCC but was optimized for performance and stability by , 15. AlkSerum bilirubin concentration was determined using Roche/Hitachi cobas c systems automatically and total bilirubin was determined by Diazo method , while dt-test. Correlation among the investigated parameters was tested by curves and regression using linear regression to test departure from linearity with runs test. These analyses were carried out using computer statistics Prism 3.0 Package . The minimum level of statistical significance was set at p < 0.05, 0.01, or 0.001.The data were expressed as mean ± SE. The results were analyzed statistically using column statistics and one  P. falciparum 11 (16.4%) had low intensity of infecting low 1+ (1–10/100 fields), 14 (20.9%) had mild intensity of infection 2+ (11–100/100 fields), 23 (34.3%) had moderate intensity of infection 3+ (1–10/one field), and 19 (28.4%) had high intensity of infection 4+ (>10/one field)  . There wsubjects .  Table  P. falciparum affected patients compared to the healthy subjects. This finding agrees with previous reports [ P. falciparum affected patients was 13.7 gm/dL. This value was more than that observed in our study. Reduced hemoglobin in malaria may be attributed to the increase of breakdown red blood cells by the parasites [ P. falciparum in Aden, Yemen.In this study, hemoglobin (Hb) and packed cell volume (PCV) decreased in reports , 19. The reports , hemogloarasites . Accordiarasites  as contaarasites , it was  falciparum malaria patients compared to the healthy subjects was observed in our study. This was in concordance with other studies [ P. falciparum as compared to those not infected . Reduction in the counts of lymphocytes was observed in some studies [Lower mean value for leukocyte count in the studies . Similar studies , who rep studies . The dec studies . P. falciparum as compared to those not infected. These findings are in agreement with previous reports [The present study reports a significant increase in neutrophil level of individuals infected with reports , 25. Inc reports , 27, 28. P. falciparum malaria. The increased serum levels of hepatic enzymes, transaminases (AST and ALT), and ALP are the biomarkers of liver disorders. Our results are consistent with other studies which reported that majority of the patients show elevation in serum activities  indicating liver damage [The results reported in this study show some significant increases in activities enzymes aspartate transaminase (AST), alanine transaminase (ALT), and alkaline phosphatase (ALP) among patients withr damage , 30. Ther damage  reportedr damage . In thisr damage  suggester damage , 35. falciparum malaria infection is associated with an increase in serum activity of aspartate and alanine aminotransferases and alkaline phosphatase thus indicating that the infection is associated with acute liver injury.Hemoglobin, platelets count, lymphopenia, tWBC, and neutrophilia were seen associated with mild, moderate, and severe parasitaemia infection. Acute


Plasmodium falciparum circumsporozoite protein (CSP) from the 3D7 reference strain and has shown modest efficacy against clinical disease in African children. It remains unclear what aspect(s) of the immune response elicited by this vaccine are protective. The goals of this study were to measure diversity in immunogenic regions of CSP, and to identify associations between polymorphism in CSP and the risk of P. falciparum infection and clinical disease. The present study includes data and samples from a prospective cohort study designed to measure incidence of malaria infection and disease in children in Bandiagara, Mali. A total of 769 parasite-positive blood samples corresponding to both acute clinical malaria episodes and asymptomatic infections experienced by 100 children were included in the study. Non-synonymous SNP data were generated by 454 sequencing for the T-cell epitopes, and repeat length data were generated for the B-cell epitopes of the cs gene. Cox proportional hazards models were used to determine the effect of sequence variation in consecutive infections occurring within individuals on the time to new infection and new clinical malaria episode. Diversity in the T-cell epitope-encoding regions Th2R and Th3R remained stable throughout seasons, between age groups and between clinical and asymptomatic infections with the exception of a higher proportion of 3D7 haplotypes found in the oldest age group. No associations between sequence variation and hazard of infection or clinical malaria were detected. The lack of association between sequence variation and hazard of infection or clinical malaria suggests that naturally acquired immunity to CSP may not be allele-specific.The malaria vaccine candidate RTS,S/AS01 is based on immunogenic regions of  Plasmodium falciparum blood stage antigens found that immune responses to these antigens may be allele-specific, and a field trial of a vaccine based on one of these antigens reported allele-specific efficacy To date, malaria vaccine development and testing has generally not been informed by molecular epidemiologic evaluations of how genetic diversity in vaccine antigens in parasite populations may affect vaccine efficacy. For example, vaccines that confer allele-specific protection may not be effective in a parasite population in which the vaccine allele is rare, and may create a selective advantage favoring non-vaccine alleles, compromising vaccine efficacy P. falciparum circumsporozoite protein (CSP), encoded by the cs gene. The cs gene is polymorphic, with diversity in regions that encode epitopes recognized by the human immune system. The central repeat region of the cs gene contains tetrameric repeats that vary in both the sequence and number of tetramers. This region codes for epitopes recognized by anti-CSP antibodies cs gene, Th2R and Th3R, encode epitopes that are recognized by CD8+ and CD4+ T-cells The malaria vaccine RTS,S/AS01 targets specific immunogenic epitopes of the Follow-up studies to Phase 2 trials of the vaccine evaluated evidence of selection of non-vaccine strains in vaccinated vs. non-vaccinated study participants. In a Phase 2 trial of RTS,S in children in Mozambique no evidence of selection favoring non-vaccine strains was found cs gene which encodes the B-cell epitopes of CSP and which is also included in the vaccine, was not considered.Both of these studies used direct sequencing to detect polymorphism in the regions coding for the T-cell epitopes, Th2R and Th3R, and excluded samples that could not be resolved into predominant alleles from the analysis. Moreover, diversity in the central repeat region of the P. falciparum antigens, were associated with a higher risk of clinical malaria To design more effective malaria vaccines and to help interpret efficacy data from vaccine trials, an understanding of the dynamics of polymorphism in vaccine antigens and the factors that are driving that polymorphism is necessary. With the aid of next generation sequencing to help circumvent some of the issues encountered by previous studies of genetic diversity in CSP, this study has examined both the population level and within-host dynamics of polymorphism in this vaccine antigen. We have previously shown that in consecutive infections experienced by Malian children followed in a prospective cohort study, changes at specific polymorphic sites, but not at other polymorphic sites, within two blood-stage The study was approved by the institutional review boards of the University of Sciences, Techniques and Technology Faculty of Medicine, Pharmacy and Dentistry in Bamako, Mali and the University of Maryland, Baltimore. Informed consent was obtained from all study participants or their guardians. The trial was conducted in compliance with the International Conference on Harmonization of Good Clinical Practices, the Declaration of Helsinki, and regulatory requirements of Mali.falciparum malaria is intense with the peak coinciding with the rainy season from July–October. Children aged less than 10 years experience, on average, two clinical episodes a year, and the prevalence of parasitemia at the beginning of the study before the onset of malaria transmission was 17% The study was conducted in Bandiagara, a rural town of approximately 13,000 inhabitants located in central Mali. Transmission of A complete population census was conducted in Bandiagara before study initiation. Study participants were sampled in proportion to the population size in each of the eight districts comprising the town of Bandiagara. Study subjects were aged ≥3 months to 20 years. Recruitment of study subjects took place in randomly selected households until the target number of subjects in each age group was achieved.P. falciparum asexual parasites and symptoms consistent with malaria including, fever, anemia, headache, body aches, cough, diarrhea, or abdominal pain, in the absence of any other obvious cause of symptoms. Infections were defined as the presence of falciparum parasites in the blood, with or without symptoms The study was conducted prospectively during the years 1999, 2000, and 2001. From July to January of each year, blood samples were collected on 3 MM Whatman filter paper monthly and at every episode of clinical malaria. Clinical malaria episodes were detected both by passive surveillance through provision of around-the-clock free clinical care, as well as by active weekly follow-up of the children in the study by physicians working at the study site. Clinical episodes were defined as blood smears positive for One hundred children with at least two years of follow up during the malaria incidence study were chosen GTTGAGGCCTTTTCCAGGAATACCAG and GTACAACTCAAACTAAGATGTGTTC. These primers were designed to amplify the region of the cs gene that contained both the repeat region and the Th regions. Primary PCR conditions are as follows: 30 cycles of 95°C for 30 s, 52°C for 30 s, 72°C for 1 min. The secondary forward and reverse PCR primers for the repeat region were TGGGAAACAGGAAAATTGG and GCACTGTTGGCATTAGCATTT. Secondary PCR conditions for the repeat regions and ThRs were 30 cycles of 95°C for 30 s, 55°C for 30 s, 72°C for 1 min 30 s, and 25 cycles of 95°C for 30 s, 58°C for 30 s, and 72°C for 1 min respectively. PCR products were amplified using HotStar Taq . Secondary PCR primers for the ThRs contained specific adapters necessary for the emPCR CGTATCGCCTCCCTCGCGCCATCAG and CTATGCGCCTTGCCAGCCCGCTCAG. Sequence specific primers for the Th and repeat regions were previously reported in a methods paper The primary forward and reverse PCR primers were The concentration of each PCR product containing Th2R and Th3R was determined by band intensity measurements taken on the Qiaxcel capillary gel imaging system , and 100 ng of each product was then pooled. Identical barcodes were used to tag more than one sample. PCR products were separated into sixteen separate pools containing approximately 45 samples each ensuring that no pool contained more than one sample with the same barcode. Each pool was physically separated within a 454 sequencing run. Pooled PCR products were sequenced at the University of Maryland School of Medicine Genomic Resource Center at the Institute of Genome Sciences on the GS FLX Titanium 454 Platform . Sequences were aligned using gsAmplicon  software. For samples containing more than one allele at a polymorphic site, predominance was determined if the majority allele was present in 71% or more of all reads obtained for that sample. This cut-off was determined in the methods validation study performed by the same authors comparing 454 and Sanger sequencing The methods validation study established the comparability of these methods with direct sequencing as well as comparable majority/minority allele cut-off values. The results of that study indicated that 454 was more sensitive at detecting minor alleles and more accurate in the quantitation of these alleles than direct sequencing To determine the length of the repeat region, PCR products were run on a high resolution gel cartridge on a Qiaxcel capillary gel imaging system  using the OM500 analysis method capable of resolving size within 10 base pairs (bp). To validate this method, a 96-well plate containing 15 ul of repeat region PCR product amplified from 100 ng/ul 3D7 genomic DNA per well, was run using the method listed above. A systematic underestimation of 10 bp was observed in the experimental data. A 10 bp correction was therefore applied to length values obtained for PCR products from study samples.Repeat region PCR products for samples that were determined to be ‘single clone’  with respect to Th2R and Th3R, were subjected to Sanger sequencing. Once amplification was verified by gel electrophoresis, PCR Products were purified by vacuum filtration in Excela Pure  96-well plates. Purified PCR product was then sequenced on an ABI3730 xl at the University of Maryland School of Medicine Biopolymer Lab.Sequences were viewed in Sequencher , and then copied to Transeq web software Mixed infections with respect to the ThRs were defined as samples with no clear majority allele at a given polymorphic site. Mixed infections with respect to the repeat regions were defined as samples which contained more than one clear band in high-resolution gel analysis.Fisher’s exact tests were used to compare haplotype frequencies between seasons, age groups, and between clinical and non-clinical episodes. Cox proportional hazards models were used to infer the relationship between diversity in Th2R and Th3R and the development of naturally acquired immunity in the context of symptomatic  and asymptomatic parasitemia (infection). In the clinical model, the association between changes at polymorphic sites in Th2R and Th3R which occurred between consecutive clinical episodes and the hazard of clinical disease was examined. In the infection model, the association between these changes which occurred between a clinical episode and a consecutive asymptomatic infection was examined. Intervals starting with an asymptomatic episode were included in the model if there was an intermediate time point at which the study participant was parasite-negative verified by microscopy. To account for the possibility of treatment failure and to allow for time for allele-specific antibodies to the first of two paired consecutive infections to be present by the time of the second infection, time intervals two weeks or less between consecutive episodes were excluded from the analyses. A logistic regression analysis was performed to determine the odds of a change in the predominant amino acid at polymorphic residues in intervals including an asymptomatic episode followed by a symptomatic one, to intervals including consecutive asymptomatic episodes. Age and time between consecutive intervals were included as covariates in this model.Of the 769 parasite-positive samples, 63 were missing or did not have enough material for successful PCR amplification. With respect to the Th regions, DNA was successfully amplified and sequenced from 684 (97%) of the samples. Repeat region PCR products and fragment length data were successfully generated for 504 (74%) of the samples. Of these 504 samples, 157 were successfully sequenced via Sanger sequencing.A total of 31 and 14 nonsynonymous SNPs were detected by 454 sequencing with an average coverage of ∼600x  in Th2R and Th3R, respectively . There wThe number of unique haplotypes found in Th2R and Th3R respectively was 87 and 23 . GenbankFor the purposes of this study, polyclonal infections were defined as samples that contained no clear majority allele . With respect to Th2R, 34.9% of samples were mixed, and with respect to Th3R, 29.8% of samples were mixed. Haplotype distributions did not vary significantly by age group, study year, or presence of clinical symptom for either Th2R or Th3R, with the exception of the Th2R 3D7 haplotype, which had a significantly greater prevalence in the oldest age group compared to the youngest age group p<0.03,  and 2.The prevalence of different repeat region sizes did not vary significantly by age group, study year, or presence of clinical symptoms . The mosAmong the 157 samples for which Sanger sequencing data were available, 19 unique haplotypes were detected, of which six were detected only once in the three year study period. Genbank accession numbers for the nucleotide sequence of these haplotypes are JQ868451–JQ868469. The majority of samples (67.5%) had one of three most prevalent haplotypes . When SaThere was no significant change in hazard of infection or clinical disease when a change occurred at any of the 17 polymorphic amino acid positions identified in Th2R and Th3R, or when a change occurred in the size of the repeat region  and 6. TThere was no significant difference in the odds of a change occurring between an asymptomatic episode followed by a symptomatic one compared to consecutive asymptomatic episodes, taking into account age and length of interval between episodes, for either the Th regions or the repeat region . Four ofMolecular epidemiological studies are an important tool for evaluating the potential of polymorphic malaria vaccine antigens to provide strain-transcending protective efficacy By using 454, this study was able to include samples containing more than one parasite clone, circumventing a problem that previous studies evaluating the allele-specificity of immunity to CSP have faced. Complete sequence reads were available for each parasite type detected by 454 so haplotypes could be constructed. In 684 samples, 87 haplotypes were found in Th2R, whereas in earlier studies using Sanger sequencing on samples from other West African settings with similar malaria epidemiology, 24 haplotypes were found in 44 Gambian samples Repeat region size determination was very accurate, with 94% agreement between lengths obtained by direct sequencing and those determined by high-resolution gel analysis. Using this method made it possible to examine the association between repeat region size and increased risk of infection and disease. Although the number of samples successfully sequenced was limited, results from one-third of parasite positive samples showed that 67% of successfully sequenced samples had one of three haplotypes with different lengths. This finding suggests that length polymorphism may serve as a reasonable surrogate for haplotype in the Cox and logistic regression models.This study had some limitations which may have affected the results. Due to a large degree of polymorphism in the ThRs and a limited sample size, smaller changes in the hazard ratios between consecutive episodes in which there was a change versus no change for both clinical disease and infection could have been missed. For seven of the 23 polymorphic sites that were modeled there may not have been enough observations to fully examine associations with infection and disease. Additionally, effects of sequence variation on hazard of clinical disease or infection were not examined with respect to the repeat region due to the limited number of sequences generated. Finally, determination of polyclonal infections for the repeat region was done by capillary gel analysis, which may have been less sensitive than sequence analysis.cs gene being under diversifying selection The large number of SNPs and haplotypes in our study is consistent with this region of the falciparum parasites. Combined molecular-immuno-epidemiological studies may help to assess these possibilities.If the diversity in immunogenic regions in CSP is not the target of allele-specific immune responses, the question of what is driving this diversity remains to be answered. It has been proposed that diversity in the repeat region might have evolved as an immune evasion mechanism by the parasite in which the host mounts a non-protective immune response against this region while the parasite escapes into the liver


Entamoeba histolytica (E. histolytica) usually asymptomatically colonizes the human intestine. In the minority of the cases, the parasite evades from the gut and can induce severe symptoms like colitis or amebic liver abscess (ALA). Interestingly, ALA predominates in adult men despite a higher prevalence of the parasite in women. The present study aimed to identify characteristic serum markers in a unique cohort of clearly defined asymptomatically infected E. histolytica individuals in comparison to patients with an E. histolytica liver manifestation of both sex.The protozoan parasite E. histolytica carriers (AC) (n = 44), and healthy E. dispar-infected controls (n = 24) out of an amebiasis endemic area. E. histolytica-specific immunoglobulin G (IgG) and the IgG subclasses against proteinaceous and non-proteinaceous amebic antigens were measured by ELISA. Serum cytokine and chemokine levels were investigated using a flow cytometry bead-based multiplex immunoassay.The following study groups were investigated: ALA patients (n = 38), healthy asymptomatic E. histolytica-specific titers of IgG and all IgG subclasses as well as IgA. IgG and IgG2 titers against the glycolipid E. histolytica lipophosphoglycan were highest in ALA patients. As in ALA patients, high cytokine levels of interleukin (IL-) 4 were detected in AC compared to E. dispar infected individuals, while IL-6 was exclusively elevated in ALA patients. IL-10 was lower in AC compared to ALA patients. Equal serum levels of CCL2 were found in all study groups but ALA patients showed decreased levels of CCL3. Sex dependent analysis of the data indicated significantly higher IgG and IgG1 titers in female AC compared to male AC. CCL2, the chemokine involved in immunopathology in the mouse model for the disease, was higher in male AC compared to female AC.The IgG results revealed that not only ALA patients, but also AC, developed high In this study we characterize for the first time an asymptomatic carrier stage in amebiasis that is associated with a significant immune reaction and provide immunological markers that might give first hints towards an understanding of immune mechanisms underlying the control or development of invasive amebiasis.The online version of this article (doi:10.1186/s12879-014-0621-1) contains supplementary material, which is available to authorized users. Entamoeba histolytica (E. histolytica) is the causative agent of invasive amebiasis, a disease that represents a major health problem in subtropical and tropical areas as well as in returnees from amebiasis-endemic areas . The. TheEntaic areas . Only inic areas -6]. The. TheEntaE. histolytica could represent an important group enabling the study of immune responses that are critical to the outcome of an infection. However, the identification of such individuals requires a diagnostic tool that can distinguish E. histolytica gut infections from infections with the morphologically identical but harmless intestinal parasite Entamoeba dispar (E. dispar), which is widely distributed in regions endemic for E. histolytica . Bas. BasE. hnt study ,[9].E. histolytica, and whether these responses differ from those of subjects with ALA. Invasive amebiasis is associated with the development of high anti-amebic immunoglobulin G (IgG) titers. For diagnostic purposes, the usage of a soluble amebic protein (SAP) preparation reveals high sensitivity and specificity . De. DeE. hipatients . TherefoE. histolytica trophozoites . Ig. Ig14]. hozoites ,20]..E. histoIn addition to the humoral immune response, serum levels of mediators of cellular immunity may also be suitable for elucidating immune mechanisms underlying the outcome of an infection. Apart from the pro- and anti-inflammatory cytokines, chemokines involved in innate immune mechanisms, especially the C-C chemokine ligands (CCL)2 and CCL3, are of interest. CCL2, formerly known as monocyte chemotactic protein (MCP-1), is critically involved in the immunopathology mediated by inflammatory monocytes in many human diseases and murine models for human diseases , includiE. histolytica: asymptomatic carriers of E. histolytica and ALA patients. In addition, we also analyzed the results with respect to the sex of the individuals.In the present study, we examined serum immune markers associated with resistance and susceptibility in two unique cohorts of individuals infected with E. histolytica infections in an area in central Vietnam that is endemic for amebiasis . Se. SeE. hiThe study was approved by the Scientific Council of Education, Training, and Ethics of Hué Medical School, Vietnam, on September 11, 1998, a written informed consent for participation was obtained from participants.E. histolytica trophozoites (HM-1:IMSS) following several steps of freezing and thawing , bu, buE. hiE. histolytica-specific antibody response in AC has been reported earlier. However, in one study, PCR confirmation of the parasite was not available at that time and the study group was comparatively small . In. InE. hiic lysis . Anotherhozoites ,37]. Ta. TaE. hiEhLPPG glycolipid, which is abundant on the surface of E. histolytica trophozoites and consists of carbohydrates and lipids ,. Co. CoE. dind STAT3 . Most remebiasis . In a motolytica .E. dispar groups revealed several interesting aspects. First, the three groups had equivalently high levels of CCL2, but high serum levels of CCL2 had also been shown in other healthy control groups . Se. SeE. dil groups . Becausel groups , an addi females ,49]. Ho. HoE. diE. dispar subjects and a strong tendency (p = 0.057) towards a lower CCL3 expression level compared with AC, which suggests that activated neutrophils may play a role in the control of E. histolytica gut infection. This notion is supported by in vitro and in vivo studies that show that activated human or murine neutrophils exhibit amebicidal activity ..E. dispaE. histolytica IgG1 than male AC, which suggests that resistance to ALA may be associated with complement-mediated mechanisms. The predisposition of males towards immunopathology via increased CCL2 levels during intestinal E. histolytica infection may reflect their greater tendency to recruit inflammatory monocytes.In summary, the present study supports the notion that the strong antibody responses found in AC reflect intense interactions between the host immune system and the invading pathogen in the absence of clinical symptoms. In addition, sex-related analyses revealed that female AC subjects expressed higher levels of anti-E. histolytica infections, thus overcoming deficiencies of many epidemiological studies in the past and enables and justifies state-of-the-art immunological investigations. Performing respective immunological studies on a disease like amebiasis, that offers such clearly distinguishable patient groups, could give interesting clues about the outcome of the disease amebiasis, but also on mechanisms determining sex-specific immunity in a wide spectrum of diseases.Astonishing little is known about the immune response underlying invasive amebiasis. This includes all stages of infection, ranging from asymptomatic colonization up to the invasive stages like amebic colitis or amebic liver abscess. In addition, the pronounced sex bias in the outcome of amebic liver abscess is nearly uninvestigated although sex dependent differences in susceptibility and resistance to many infections become unquestionably an increasing field of interest ,53]. No. No53].


Plasmodium falciparum malaria has been the holy grail of natural infection studies, and sought after as an endpoint for malaria vaccine trials. An in vitro assay that provides an accurate and precise assessment of protective immunity to malaria would make smaller, short-duration studies feasible, rather than the currently powered study designs that use morbidity or mortality as outcomes. Such a biomarker would be especially desirable in situations where malaria control measures that result in decreases in clinical endpoints and putatively waning protective immunity have been implemented. In an article published in BMC Medicine, Osier and colleagues addressed this problem, and demonstrated that antibodies promoting opsonic phagocytosis of merozoites provide a functional link between antigen-specific responses and protection. Understanding the mechanisms conferring protection against malaria not only improves our knowledge of basic human immunology, but promises to help in the design of an effective malaria vaccine.Determining an immunologic correlate of protection against http://www.biomedcentral.com/content/pdf/1741-7015-12-108.pdf.Please see related article:  One of the reasons that malaria vaccine development is so challenging is the enormous complexity of the host–parasite interaction. The parasite itself has over 5,000 open reading frames, some of which are differentially expressed during the numerous distinct life-cycle stages within the human host. Even within the cycling erythrocyte stage that can create the febrile disease, the parasite expresses different antigens as an invading merozoite, as a growing trophozoite, and as an organizing schizont. This remarkable life-cycle complexity, coupled with an almost unequalled degree of antigenic variability within the global population of parasites, allows the parasite to evade many actions of the host’s immune system.partial immunity – is thought to involve both cellular- and antibody-mediated mechanisms. However, a dominant role for anti-malarial antibodies was demonstrated most clearly over five decades ago by the immediate and dramatic clinical and parasitological improvement of Thai children transfused with immunoglobulin from endemic adults [Thus, unlike many other infectious diseases, survival of one or more bouts of febrile malaria disease does not provide sterile protection against further disease or further infection. Although still only partially understood, repeated exposure does change the immune balance between the parasite and its host. Because children in endemic areas age in the context of repeated infection, they gradually acquire the abilities both to control parasitemia and to resist being clinically ill -3. This c adults .in vitro methods to evaluate the functionality of antibodies to the blood-stage parasite are the growth inhibition assay (GIA), which measures the ability of antibodies to slow the expansion of P. falciparum parasites cultured in human erythrocytes in the incubator [in vivo, responses include not just the antibodies themselves but the other immune cells with which they interact, thus making this assay more biologically relevant. This new functional assay measures the ability of antibodies to opsonize freshly isolated live merozoites for phagocytosis by macrophages in short-term culture. The authors have carefully characterized this assay, and found that it works with both freshly isolated macrophages within lymphocyte mixtures and with an isolated macrophage cell line. It appears readily reproducible to any laboratory with cell and malaria culture capabilities and a flow cytometer.Antibody generation has therefore remained an important goal for blood-stage malaria vaccination efforts. Simple anti-repeat region antibodies correlate fairly well with the protection afforded by the most advanced malaria vaccine candidate to date, GlaxoSmithKline’s RTS,S, but the paradoxically consistent presence of individuals who are protected from challenge with barely detectable antibody responses makes this a less than ideal predictive biomarker  for thisncubator , and thencubator . In theincubator  have desP = 0.041). The addition of this novel functional assay as a biomarker seems a logical step towards a better understanding of the various specificities involved in the development of protective immunity to malaria.The most compelling evidence presented by Osier and colleagues are the correlations within the context of two independent longitudinal cohort studies conducted on the coast in Kenya. The naturally acquired responses measured by this OPA correlated with the ability to resist clinical illness in both groups of children residing in this malaria endemic area. Activity in OPA significantly correlated with IgG ELISA but had a weak correlation with GIA  of merozoites between schizont rupture and re-invasion, although the short duration of the assay may functionally mimic this. Because it is an assay that utilizes a whole, living merozoite, its applicability to screen for efficacy of a single-allele, single-antigen vaccine is difficult to determine. Thus, this tool should be championed as a functional assay that, perhaps through future antigen add-back or blocking experiments, may help dissect the diverse antibody responses developing in sequentially exposed individuals to determine which of them are functionally important in resisting disease. This work also sets the stage to develop similar opsonization assays against the infected red blood cell, which is presented to the host immune responses for a considerably longer duration.Although the correlation between naturally acquired partial protection and the results of OPA is very suggestive, the link predicting protection against malaria induced by a vaccine presenting a limited number of antigens remains untested. It is possible that a vaccine candidate might induce antibodies that perform well in the OPA, yet fail to predict the outcome of a challenge infection, as has been the case with GIA and anti-malarial antibodies in general. Caution in generalizing to the in vitro assay that can, if not predict the efficacy of a vaccine, at least correlate well with it. One of the perennial frustrations of those seeking to understand the interaction of the malaria parasite with the human immune system is its enormous complexity and the limitations in the tools currently available with which to dissect that complexity. Whether for vaccine development or for understanding the basic immunology that underlies the gradual acquisition of partial resistance to the disease manifestations of malaria parasitemia, the technical advance described in the current paper by Osier et al. [In the headlong race to develop potential malaria vaccine candidates, the as yet undiscovered ‘golden chalice’ remains an r et al.  represen