Plasmodium ovale spp. has been neglected by and large from research and has received only little scientific attention during the past decades. Ovale malaria is considered to feature relapses by liver hypnozoites although scientific evidence for this paradigm is scarce.Malaria caused by Plasmodium malariae was diagnosed by microscopy and the patient was treated accordingly with a full course of supervised artemether–lumefantrine. He was discharged in good clinical condition with a negative blood smear. One month after initial diagnosis, he returned complaining of fever. Thick blood smear was positive again for malaria parasites, which were confirmed as P. ovale wallikeri by PCR. Retrospective analysis revealed the identical Plasmodium spp. in the initial blood samples. Molecular analysis of various gene loci  gave identical results providing further evidence for relapse by an identical parasite genotype. Consecutively, the patient was retreated with artemether–lumefantrine and received a regimen of primaquine according to WHO guidelines.Here, the case of a 16-year-old male, who presented with fevers to the outpatient department in Vienna, Austria, after travelling to Uganda and Papua New Guinea is described. Infection with P. ovale spp. is rare. The presented case provides convincing confirmation for the relapse paradigm based on re-appearing parasitaemia following supervised treatment in a non-endemic region with a parasite strain of identical genotype.Conclusive evidence for relapses with  Plasmodium ovale malaria has been neglected largely from research in the past decades with the primary focus being laid on falciparum and to a lesser extent vivax malaria. Even though evidence is scarce, P. ovale is considered a relapsing Plasmodium spp. by analogy to Plasmodium vivax for which evidence for liver hypnozoites is well-established [ablished .P. ovale hypnozoites was, however, never firmly established nor has a clinical trial on the nature of such relapses been performed [P. ovale relapses has derived from observations from malaria infection therapy for neurosyphilis in the first half of the 20th century or from case reports. Further evidence making use of molecular methods is largely missing. Astonishingly, as recently described by a systematic review, from the first description of the species in 1922 until 2015 less than 20 case reports have been published on potential relapses of P. ovale spp. [P. ovale is, therefore, warranted not least to provide evidence for current guidelines for anti-hypnozoitocidal treatment with primaquine.In general, a recurrence of parasites after initial treatment can be due to recrudescence of a not completely cleared bloodstream infection, to a new infection or to a relapse originating from dormant liver stages. The existence of erformed . Most of® Flu, Cepheid) was negative for Influenza A and Influenza B, as well as ELISAs for CMV and EBV. An aerobic blood culture was positive for Staphylococcus hominis after 20.4 h. This was not confirmed by other simultaneously drawn blood culture sets and was thus regarded as contamination. Physical examination was normal apart from an enlarged sub-angular lymph node on the right side. The anamnesis revealed that the patient had returned from a trip of 1 ½ months in duration, to Uganda, Papua New Guinea and Singapore, 3 months before the onset of symptoms. During this stay and until 3 weeks after returning home, he took continuous malaria prophylaxis consisting of mefloquine (Lariam®) once per week. He did not report any associated complaints. Eight months before, he had stayed at the Philippines for 10 days.A 16-year-old male patient presented with a 5-day history of recurrent fever attacks up to 39.5 °C and with night sweats and arthralgia to the outpatient department (OPD) of the infectious diseases ward of the General Hospital of Vienna. Laboratory examination indicated a moderately increased CRP (1.57 mg/dL) with a normal white blood count, thrombocytopaenia of 105 g/L, a moderately raised total bilirubin of 2.33 mg/dL in addition to moderately increased LDH of 277 U/L. Differential blood count showed monocytosis and atypical lymphocytes. A rapid diagnostic influenza test  was initiated according to the manufacturer’s recommendations and following WHO treatment guidelines. On the first day of treatment body temperature increased to a maximum of 39.8 °C, but the general health status improved subsequently. A thick blood smear on the last day of treatment was negative and the patient was discharged in good condition. A follow-up examination was planned for 1 week after admission to which the patient, however, did not present.Thick and thin blood smears were performed and ® and samples from the first and second fever episode were sent for species specific PCR diagnosis [P. ovale wallikeri mono-infection at both time points. Further analyses were initiated with the aim to identify whether the second attack was a relapse of the same parasite isolate or a prolonged latency of another P. ovale wallikeri parasite obtained during the preceding trips.One month later, the patient returned to the outpatient department with reported fever. He had not travelled abroad since the initial malaria diagnosis. A thick blood smear showed a recurrence of parasites with a very low parasitaemia. The patient was retreated with Riametiagnosis . PCR fro18S rRNA gene was analysed to identify Plasmodium spp. (Primers rOVA1WC/rOVA2WC) [P. ovale curtisi (rOVA1/rOVA2) [P. ovale wallikeri (rOVA1v/rOVA2v) [porbp2 and potra genes were analysed.Molecular analysis of several gene loci was conducted to evaluate both isolates. Initially, a fragment of the nuclear rOVA2WC) . The pos1/rOVA2)  and P. o/rOVA2v) . To examporbp2 [potra [P. ovale wallikeri isolate 17T1554/17T186R. porbp2, MG255222–MG255223—P. ovale wallikeri isolate 17T1554/17T186R SSU rRNA gene, partial sequence).Examined sequences of the SSU rRNA, porbp2 , 7 and p2 [potra  genes reAccordingly, the patient’s glucose-6-phosphate dehydrogenase (G6PD) status was tested and showed normal enzyme function so that a 14 days course of primaquine was initiated . During Plasmodium by microscopy of blood smears is still the gold standard for malaria diagnosis and provides a fast and valid diagnostic tool when performed by experienced slide readers. Since the introduction of species-specific diagnosis using PCR it has, however, become more and more evident that a considerable number of microscopically retrieved results are inaccurate, especially concerning species distinction in non-falciparum and mixed Plasmodium infections [Detection of fections –12. The P. ovale malaria. To current knowledge, no other case with subsequent molecular analysis of primary and recurring infection has been reported from a non-endemic area [P. ovale parasite originally acquired at the same time causing the second episode is negligible as the obtained sequence data strongly suggest the presence of the identical malaria parasite during the initial and the recurring episodes. Here, sample amount was not sufficient but in upcoming studies whole genome sequencing is recommended to prove identical genotypes. More difficult is a distinction between recrudescence and relapse. However, the blood-stage medication was taken under observation and there are no known specific drug resistance mechanisms for P. ovale spp. against artemether or lumefantrine, thus a recrudescence seems very unlikely.This case adds valuable information to the scarce evidence available in the literature on the relapse hypothesis for mic area , 13. HerP. vivax infections than as monotherapy. Based on this, 8-aminoquinolines were further developed, but their effectiveness against relapsing P. ovale infections was merely presumed [P. ovale dormancies in human liver cells, if only for a limited time, and raise hope that more light will be shed on the relapse phenomenon [Anti-hypnozoite treatment with 8-aminoquinolines was introduced based on the observation that quinine therapy was more efficacious in combination with the 8-aminoquinoline pamaquine for the treatment of relapsing presumed . Models enomenon , 16.P. ovale infection within the 8 months of follow up was significantly lower in the group that received primaquine. The same was true for P. vivax. For Plasmodium falciparum or P. malariae, however, no significant association of this kind could be observed. These results support the hypothesis of a quiescent parasite stage that is affected by primaquine. Yet it is to note that this was a limited sample size and no analyses were made regarding whether the observed episodes were following a previous P. ovale infection or not.In 2015, Robinson et al. conducted a randomized double-blind placebo-controlled trial comparing blood stage drugs plus primaquine to blood stage drugs plus placebo in Papua New Guinean children . The incPlasmodium spp., respectively [Plasmodium spp., dormant stages were also reported in the epidermis, brain, spleen, kidneys and lungs [Interestingly, recent studies doubt the exclusive connection between liver dormancies and tertian malaria and suggest other havens of dormancies and a relapse potential of all human infective ectively , 18, 19.nd lungs , 21. Furnd lungs , 23.P. ovale. Still, more evaluations of this kind are needed and the causal relationship between relapse and liver dormancies remains to be further explored.This case report provides an important piece in the puzzle to substantiate the relapsing properties of


The performance of different malaria rapid diagnostic tests (RDT) may be influenced by transmission intensity and by the length of time each test requires to become negative after treatment and patient’s recovery.Results of three RDTs (two HRP2 and one pLDH antigen-based tests) were compared to blood smear microscopy (the gold standard method) in children under 5 years of age living in a high versus low malaria intensity setting in southwestern Uganda. In each setting, 212 children, who tested positive by at least one RDT and by microscopy, were treated with artemether-lumefantrine. RDTs and microscopy were then repeated at fixed intervals to estimate each test’s time to negativity after treatment and patient recovery.In the two settings, sensitivities ranged from 98.4 to 99.2 % for the HRP2 tests and 94.7 to 96.1 % for the pLDH test. Specificities were 98.9 and 98.8 % for the HRP2 tests and 99.7 % for the pLDH test in the low-transmission setting and 79.7, 80.7 and 93.9 %, respectively, in the high-transmission setting. Median time to become negative was 35–42 or more days for the HRP2 tests and 2 days for the pLDH test.versus missing malaria cases.High transmission contexts and a long time to become negative resulted in considerably reduced specificities for the HRP2 tests. Choice of RDT for low- versus high-transmission settings should balance risks and benefits of over-treatment Trial registration: Registry number at ClinicalTrial.gov: NCT01325974The online version of this article (doi:10.1186/s12936-016-1529-6) contains supplementary material, which is available to authorized users. Diagnosis of malaria based only on clinical grounds leads to significant overestimates of malaria illnesses even when diagnosis is carried out by experienced clinicians , 2. MisdThe number of different RDTs on the market has increased considerably over the past few years. In 2008, WHO launched the Product Testing Programme, which evaluates performance of commercialized malaria RDTs against a standardized panel of laboratory-prepared specimens. This programme, which has already completed its fifth round of testing, allows for product comparisons, encourages producers to improve their tests and, in the long run, promotes product quality. WHO acknowledges however, that its results cannot predict test performance in the field, where other factors such as malaria antigen persistence in the blood and malaria endemicity can influence RDT performance .Plasmodium lactate dehydrogenase (pLDH), while others are designed to detect both antigens in a single test.Most malaria RDTs are designed to detect a single parasite antigen, the histidine-rich protein 2 (HRP2) or the Plasmodium falciparum species; detection of this protein can therefore confirm only P. falciparum infections. Many of the HRP2-based RDTs show very high scores in the WHO evaluation [HRP2 is a protein produced only by the aluation . For insaluation –10. HowePlasmodium species; RDTs based on this antigen can therefore detect all Plasmodium species but are generally less sensitive than HRP2 tests. Their main advantage is that pLDH is rapidly eliminated from the blood stream after treatment, and therefore pLDH-based RDTs returns a negative result within a few days after treatment.The pLDH is a parasite protein produced by all The objective of this study was to explore whether and how much three malaria RDTs vary, in terms of sensitivity, specificity and time to become negative, based on transmission setting. The three RDTs chosen were among the highest scoring tests in the WHO Round 3 malaria test performance evaluation  , and Kazo, located 80 km north of Mbarara municipality, where malaria prevalence was ten-fold higher (40–60 %) as reported in a previous study [Plasmodium falciparum is almost the only species documented. Malaria constitutes one of the major causes of illness in the area.The Greater Mbarara district is located between 130 m and 1500 m altitude above sea level and its vegetation is a mixture of bush and short grass. The climate is tropical with a bi-modal rainfall pattern averaging 1200 mm per annum. The rainfall season falls between mid-August to December and mid-February to May. Malaria transmission is on-going throughout the year, with two seasonal peaks occurring 2 months after the periods of heavy rainfall. The sample size was calculated to respond to the main study objective, the time to become negative of RDTs, and was based on the expected proportions of RDTs that remained positive at each follow-up day. It was estimated that 184 patients were needed to achieve the accuracy of 8 % around a proportion of 50 % positive RDTs and of 3 % around proportions of 5 or 95 %. By expecting 15 % of patients not being positive for all RDTs, lost to follow up or excluded from the primary analysis, 212 patients were estimated to be included in each study site.P. falciparum infection were included until the sample size for the time to become negative analysis was achieved. For the specificity analysis, the number of patients with a negative microscopy depended of the intensity of malaria transmission. A negative/positive microscopy ratio of around 1:1 and of at least 5:1 was expected in the high and low transmission settings respectively.For the sensitivity analysis all patients with a positive microscopy with a The study recruited consecutively all children who presented to any of the participating health centres, were under 5 years of age, weighed 5 kg or more, and either had fever ≥37.5 °C or whose parent reported fever within the previous 2 days. Blood samples obtained by finger prick from children whose parent or guardian provided written consent were tested in parallel at the corresponding study health centre with the three different RDTs and with microscopy. Patients recruited at this stage formed the population sample for the RDT performance analysis.P. falciparum mono-infection  confirmed by microscopy, and having not taken any anti-malarial drug within the previous 2 weeks (as reported by the parent). Participants stayed with a parent or guardian in an observation room of the health centre for 3 days (day 0–day 2) to receive a full course of artemether-lumefantrine  under the supervision of study personnel. When treatment intake was completed, participants were sent home. During their stay at the health centre and at follow-up visits on a fixed schedule  patients received a clinical check-up and malaria testing using all three RDTs and microscopy. If an RDT was negative at a given visit, that test was not repeated during subsequent visits. Follow-up was discontinued as soon as all RDTs were negative, or at a maximum of 42 days.At each site, a sub-set of 212 children was selected for the time to become negative analysis. Inclusion criteria were: positivity for at least one RDT, having a Patients with recurrent parasitaemia during follow-up were treated with artesunate-amodiaquine . Patients who did not appear for a follow-up visit after day 3 (when they went home from the centre) and for whom it was not possible to recover any additional information were considered lost to follow-up.The following RDTs were evaluated: SD Bioline Malaria Antigen P.f (HRP2) , CareStart Malaria HRP2 (Pf)  and CareStart Malaria pLDH (PAN) . RDTs were performed according to manufacturer’s instructions. All RDTs were read by two blinded, independent readers. The first reading was done at 15 min for the SD Bioline test and 20 min for the two CareStart tests. The first reader wrote the result in a paper form which was handed to the lab supervisor. The supervisor called within 5 min the second reader who wrote the result in a second paper form. The supervisor confronted the results. If results were discordant the supervisor acted as third tie-breaker reader. The third reading occurred typically within 10 min after the first reading. The lot number of each test was recorded and the storage temperature monitored.Plasmodium species was confirmed on the thin smear. Two-hundred high-power fields were read before declaring a blood slide negative.For microscopy, thick and thin blood smears were prepared on the same slide and stained with a 10 % Giemsa solution (pH 7.2) for 15 min. Reading was performed using a 100× magnification lens with oil immersion. Parasite asexual forms were counted against a threshold of 200 white blood cells (WBC); if fewer than ten parasites were seen at this threshold, counting was continued until at least 500 WBC. Parasite density was estimated based on a hypothetical leukocyte density of 8000 WBC/µL. The presence of gametocytes was recorded, although a slide with gametocytes but no asexual parasite forms was scored as negative.Double reading of all slides was performed. Slides with discordant results (positive/negative or parasite density of second reader varying by ≥50 % compared to the first reader) were read by a third reader. The parasite density result was calculated as the mean of the first and second readers, if not discordant, or as the mean of the two closest results, if a third reading was required. Blinding of microscopists to RDT results was ensured by assigning to the slides a different laboratory code, so that only the laboratory supervisor could match the microscopy result to the patient’s identification number that was used for the RDT. All study microscopists had a Kappa score ≥80 % in evaluations of microscopists conducted by the reference laboratory of the Shoklo Malaria Research Unit (Thailand) in 2010, prior to the start of this study.Data were double-entered and validated in a Voozanoo database . Data analysis was carried out using Stata version 12.1 .t test if normally distributed, and with Mann-Whitney rank-sum test if not normally distributed.Qualitative variables were assessed by percentages and 95 % confidence intervals (95 % CI). Quantitative variables were described by mean and standard deviation if normally distributed, and by median and interquartile 25 and 75 percentiles range (IQR), if not normally distributed. Comparison of continuous numeric variables was tested with Student P. falciparum (mono- or mixed species infection) were included in the analysis, while patients with an infection that did not include P. falciparum were excluded, since the HRP2 tests in the study detect only P. falciparum.Sensitivity, specificity, positive and negative predictive values, and misclassification rates were estimated for each RDT separately, with microscopy as the gold standard. All patients with RDT sensitivity and specificity were defined as the proportion of positive test results among the microscopy-positive blood smears, and the proportion of negative tests among the negative blood smears, respectively. The positive and negative predictive values were defined as the proportion of positive blood smears among the positive tests, and of negative blood smears among the negative tests, respectively. The misclassification rate was defined as the proportion of erroneous results  among the total number of tests performed.Performance comparisons between RDTs in the same site, and of the same RDT among sites, were expressed as differences with 95 % CI. McNemar’s test was used to assess statistical significance in performance among RDTs in the same site while Fisher’s exact test was used to assess statistical significance in performance of the same RDT between sites.Sensitivity of RDTs in three strata defined as low (1–1999 parasites/µL), medium  and high parasite density  were also assessed and compared. For this last analysis, results from the two sites were combined.The time to become negative was defined as the 1st day when an RDT was reported as negative. Patients with incomplete or incorrect intake of anti-malarial treatment and patients who presented recurrent parasitaemia starting from day 7  before all RDTs had become negative were excluded from the analysis. Patients lost to follow-up were censored at their last visit.The probability of a test remaining positive over time was calculated with the Kaplan–Meier survival function. The same survival analysis was carried out on three strata according to initial parasitaemia. Strata were defined as low, medium and high parasite densities (same cut-off as for RDT performance).Médecins Sans Frontières, the Faculty Research Ethical Committee and the Institutional Review Board of Mbarara University of Sciences and Technology, and the Uganda National Council for Sciences and Technology.The study protocol was approved by the Ethical Review Board of All patients were recruited after a parent or guardian provided written consent, and all patients received free health care during the entire period of study participation.Recruitment in the Mbarara municipality took place from November 2011 until the end of January 2013, an extended period that reflected the area’s low malaria transmission, and consequently, the long time needed to recruit sufficient numbers of malaria-positive patients. Patients in high-transmission Kazo were recruited from September to December 2011.A total of 4977 and 521 patients were admitted to the study in Mbarara and Kazo, respectively, of whom 4803 and 459 patients were included in the RDT performance analysis. The reasons for exclusion are listed in  and 98 (46.2 %) patients in Mbarara and Kazo, respectively, were excluded from the analysis at some point before completion of their planned visits. The most common reason for exclusion was the occurrence of a recurrent parasitaemia during follow-up, which was found in eight patients (3.8 %) in Mbarara and 89 patients (42 %) in Kazo. Other reasons are listed in the study flow chart  and very high for one patient . Specificities of all three RDTs were high in Mbarara, ranging from 98.8 to 99.8 %. In Kazo, the specificities were 79.7 % for SD Bioline HRP2 80.7 % for CareStart HPRP2 and 93.9 % for CareStart pLDH, which were significantly inferior to Mbarara, especially for the two HRP2 tests (19.2 % for SD Bioline HRP2 and 18.1 % for CareStart HPRP2). A less marked, though still significant, inferior specificity (5.8 %) was observed for the CareStart pLDH.RDT performance data in the different settings are summarized in Table The positive predictive values of the two HRP2 tests were similar in Mbarara and Kazo. The negative predictive value of the pLDH test was lower in Kazo (but not in Mbarara) compared with the HRP2 tests.Variation in RDT performance based on parasite density stratification was similar at the two study sites  in the high-transmission setting of Kazo confirmed previous studies from similar epidemiological contexts, for example, 73 % in Ghana  and 63 %The long-lasting HRP2 antigens, when combined with a high frequency of malaria infections, may lead to quasi-persistent antigen positivity in children living in high malaria transmission areas. A clear demonstration of this situation is the high number of children (86 out of 212) in Kazo that experienced a recurrent malaria infection before clearing the HRP2 antigens of the previous one. This is not the case for children living in low-transmission settings, who are much less likely to have had a recent malaria infection.Unlike specificity, the sensitivity of all three RDTs was high in both settings; however the pLDH test performed less well than the HRP2 tests. Sensitivity estimates confirm the WHO evaluations , 7 and aData of this study clearly indicate that the relatively low sensitivity of the pLDH test was observed only at low parasite density. Nevertheless, this low sensitivity is highly problematic for public health decision makers, who typically favour deploying highly sensitive tests such as the HRP2 RDTs, accepting the associated risk of unnecessarily treating uninfected patients over taking the risk of failing to detect malaria cases. Regarding specificity, the high number of false positives observed with HRP2 tests is also problematic, particularly in high transmission settings where half or more of RDTs are positive. A wrongly positive result may divert clinicians’ attention from other possible aetiologies of febrile illnesses, or may lead clinicians to mistrust the RDT result, diminishing their perceptions of the test’s usefulness. The two HPR2 tests showed much longer times to become negative (median of 35 to >42 days) than expected from most previous reports. Only one study in the Democratic Republic of Congo  reportedIn contrast, the two-day median time of pLDH antigen persistence generally seems to parallel a decline in the number of viable parasites during treatment , 17, 18.While time to become negative did not vary substantially according to the intensity of transmission, it differed based on initial parasite density: the higher the initial parasite density, the more time was required to clear parasite antigens . This fiThe study had a number of limitations. Some negative microscopy results may represent real infections with a sub-microscopic parasite density. In this case, if the RDT was positive, results may have been wrongly classified as false positive instead of true positive. Considering the high sensitivity of HRP2 tests in low parasitaemias, this circumstance may have affected a higher proportion of HRP2 test results than pLDH results.Double reading of all slides and the inspection of 200 fields before scoring a blood smear as negative were performed to ensure high quality results. Nevertheless, it may be possible that some low parasite densities went undetected and were therefore wrongly classified as negative [The PCR molecular assay is proved to be more sensitive than a well performed microscopy  and woulChildren with recurrent parasitaemia during follow-up were excluded from the time to become negative analysis. This choice had the consequence of reducing the size of the analysable patient cohort. Reduction was limited in the low-transmission setting (eight patients in Mbarara), but led to exclusion of one-third of all patients at the Kazo site (89 patients). The characterization of polymorphic markers to distinguish a re-infection from a recrudescence, a standard method in anti-malarial efficacy studies, would have allowed to censor, rather than to exclude, these children.The time to become negative for a given RDT was determined on the basis of the first negative result during follow-up, and assumed that any subsequent result with the same RDT was also negative. It may be possible that, in some few instances, a subsequent RDT could have been positive. This definition may therefore have biased estimates of time to become negative towards shorter times.The ideal malaria RDT, a test that is both highly sensitive and highly specific in all epidemiological contexts is not yet available. A choice is to be made between an HPR2 test, with the risk of overdiagnosing malaria and thereby overlooking other possible causes of fever, and using a pLDH test, which carries the risk of missing true malaria cases with low parasitaemia. Three-band RDTs, combining detection of both HRP2 and pLDH antigens, are also available. However these RDTs are not better performing than the two-band tests  and may


Plasmodium falciparum populations and multiplicity of infection (MOI) in Grande Comore 10 years after introduction of ACT.Malaria is still one of the serious public health problems in Grande Comore Island, although the number of annual cases has been greatly reduced in recent years. A better understanding of malaria parasite population diversity and transmission dynamics is critical for assessing the effectiveness of malaria control measures. The objective of this study is to investigate temporal changes in genetic diversity of P. falciparum clinical isolates were collected from the Grande Comore Island during two sampling periods . Parasite isolates were characterized for genetic diversity and complexity of infection by genotyping polymorphic regions in merozoite surface protein gene 1 (msp-1), msp-2, and msp-3 using nested PCR and DNA sequencing.A total of 232 msp-1 alleles , two msp-2 alleles (FC27 and 3D7), and two msp-3 alleles (K1 and 3D7) were detected in parasites of both sampling periods. The RO33 allele of msp-1 (84.8%), 3D7 allele of msp-2 (90.8%), and K1 allele of msp-3 (66.7%) were the predominant allelic types in isolates from 2006–2007 group. In contrast, the RO33 allele of msp-1 (63.4%), FC27 allele of msp-2 (91.1%), and 3D7 allele of msp-3 (53.5%) were the most prevalent among isolates from the 2013–2016 group. Compared with the 2006‒2007 group, polyclonal infection rates of msp-1  and msp-2  allelic types were significantly decreased in those from 2013‒2016 group. Similarly, the MOIs for both msp-1 and msp-2 were higher in P. falciparum isolates in the 2006–2007 group than those in 2013–2016 group . DNA sequencing analyses also revealed reduced numbers of distinct sequence variants in the three genes from 2006‒2007 to 2013‒2016: msp-1, from 32 to 23 (about 28% decline); msp-2 from 29 to 21 (about 28% decline), and msp-3 from 11 to 3 (about 72% decline).Three P. falciparum populations over the course of the study, suggesting a trend of decreasing malaria transmission intensity and genetic diversity in Grande Comore Island. These data provide valuable information for surveillance of P. falciparum infection and for assessing the appropriateness of the current malarial control strategies in the endemic area.The present data showed dramatic reduction in genetic diversity and MOI among Grande Comore  Plasmodium falciparum malaria had been widely distributed throughout the Union of Comoros  and posed a serious impediment to socioeconomic development historically [Malaria is a major infectious disease that led to ~ 212 million clinical cases and about 429,000 deaths worldwide in 2016 [P. falciparum parasite is the extensive genetic diversity of vaccine targets allowing parasites with mutated genes to escape from the host’s immune response [P. falciparum parasites has been widely used as an indicator of level of malaria transmission intensity in endemic regions, thus serving as a tool to evaluate the effectiveness of malaria control and intervention.To date, several malaria pre-erythrocytic (RTS/S and PfSPZ Vaccine) or erythrocytic  stage vaccines have been designed to induce immunity against the pre-erythrocytic or erythrocytic stage of the malaria parasites, respectively . Althougresponse , 8. Thusmsp-1, msp-2, and msp-3) have been widely used for characterization of parasite genetic diversity [P. falciparum parasites collected from Comoros Archipelago  using microsatellite loci [P. falciparum populations in Grande Comore were substantially different from those in other two islands (Moheli and Anjouan). Currently, no data on temporal changes in genetic diversity of P. falciparum isolates from Grande Comoros after introduction of ACT are available. Herein, the objective of this study is to investigate the dynamics of genetic diversity and multiplicity of infection (MOI) in clinical P. falciparum isolates from Grande Comore during two different periods (2006‒2007 and 2013‒2016) using polymorphic markers of msp-1, msp-2, and msp-3. The data in this study provide insights on parasite diversity and MOI after various malaria control measures.Polymorphic genetic marks, such as microsatellites and genes encoding merozoite surface proteins  and Guangzhou University of Chinese Medicine (No. 2012L0816). Blood samples were collected from children after obtaining written informed consent from their parents or legal guardians.2 with about 420,000 inhabitants (2012 estimate). A tropical hot and rainy season occurs from November to April, and a cooler dry season runs from May to October. Annual temperature ranges from 11 to 35 °C and rainfall ranges from 1000 to 3000 mm per year. Malaria transmission on this island is perennial with most of infections occurring during the rainy season. P. falciparum is the dominant malaria species, with occasional Plasmodium malariae and Plasmodium vivax infections [This study was conducted on the Grande Comore Island, Union of Comoros Fig. , that isitants 20 estimateP. falciparum positive patients visiting local healthcare facilities were collected in two different periods (March 2006–October 2007 and March 2013–December 2016). Among these samples, 118 samples from Mitsoudje Centre Hospital (50), National Malaria Centre (30), and Mitsamiouli Centre Hospital (38) were collected during March 2006–October 2007; the other 114 samples from Mitsoudje Centre Hospital (41), National Malaria Centre (24), Mitsamiouli Centre Hospital (29), and Mbeni District Health Centre (20) were collected during March 2013–December 2016. Venous blood sample of 1.0 ml was collected from each patient in an EDTA tube and stored at −20 °C until DNA extraction.A total of 232 blood samples from microscopically pfmsp-1 (block 2), pfmsp-2 (block 3), and pfmsp-3 were amplified using nest PCR, as described previously [2O, 0.5 μl of each primer (0.4 µM), 12.5 μl of Taq PCR Mast Mix (2.5 U) following the manufacturer’s instructions  on a S1000 Thermal cycler . In the primary amplification reactions, 2.0 μl of template genomic DNA were added as a temple. In the nested reaction, 0.5 μl of primary PCR product was added as a temple. The nested PCR products were separated on 2.0% agarose gel  and visualized under ultraviolet (UV) trans-illumination. A 100-bp DNA ladder was used to determine the size of PCR products . MOI of the msp-1 or msp-2 genes was calculated by averaging number of amplified bands per positive P. falciparum isolate as described previously [msp-1, msp-2, and msp-3 were directly sequenced in both directions with the primers in the secondary PCR using an ABI PRISM3730 DNA sequencer . The sequences were also used to correct the estimated molecular weight and to confirm the nature of the amplified product.Genomic DNA of each blood sample was extracted using Takara DNA Blood Mini Kit according to the manufacturer’s instructions . Extracted parasite DNA was dissolved in TE buffer  and stored in microfuge tubes at −20 °C. Segments of the eviously . An initeviously . For DNAmsp-1 or msp-2 in isolates collected between 2006–2007 and 2013–2016 were made using t test. Mann–Whitney U test was used to compare in the frequencies of the mutations and alleles of the msp-1, msp-2 and msp-3 in isolates collected between 2006–2007 and 2013–2016. P < 0.05 was considered indicative of a statistically significant difference.Statistical analysis was determined using SPSS (version 13.0) software. Comparisons of MOI of P. falciparum isolates (222/232) were successfully amplified from msp-1 block 2 region , and three allelic types  were identified from the samples (Table P < 0.01) in those from 2013–2016 group, whereas the frequencies of isolates with only K1, MAD20, or RO33 allelic types was significantly increased (P < 0.01).Approximately 98% of P. falciparum isolates had a positive PCR outcome for msp-2 block 3, including 109 samples collected during 2006–2007 and 113 samples during 2013–2016. Two allelic types (FC27 and 3D7) were identified in both two periods (Table P < 0.01). MOIs for both msp-1 (3.11) and msp-2 (2.75) were higher in P. falciparum isolates in 2006–2007 group than those in 2013–2016 group .A total of 222 msp-1 block 2 region revealed that the Grande Comore isolates contained a limited number of different tripeptide repeat units . For the K1 allelic type, the tripeptide repeat region always started with SAQ and terminated with SGT  , K1-4 (P < 0.01), K1-6 (P < 0.01), K1-10 (P < 0.01), K1-15 (P < 0.05), K1-17 (P < 0.01), and K1-18 (P < 0.05) subtypes were significantly decreased in the 2013–2017 group, whereas the K1-2 (P < 0.05), K1-5 (P < 0.01), K1-8 (P < 0.01), K1-11 (P < 0.01), K1-13 (P < 0.01), and K1-14 (P < 0.05) were significantly increased. For msp-1 MAD20 allelic types, significant decrease in MAD20-4 to MAD20-9 allelic types (P < 0.05 or P < 0.01) and significant increase in MAD20-1 to MAD20-3 types (P < 0.01) were observed in 2013–2016 group compared with those of the 2006–2007 group. Similarly, in comparison to 2006–2007 group, significantly reduced frequencies of RO33-3, RO33-5, RO33-6, RO33-8, and RO33-10 were observed in the 2013–2016 group (P < 0.01), whereas significantly increased of RO33-1 (P < 0.01), RO33-7 (P < 0.05), and RO33-11 (P < 0.01) types were detected in the 2013–2016 group.Sequence analysis of msp-2 block 3 region showed that the FC27 alleles contained a family-specific region (ADTIASGSQSSTNSASTSTTNNGESQTTTPTA), a conserved 8 amino acid region (ADTPTATE), a 12 amino acid repeating unit , and followed by a conserved region (SSGNAPNK) at the end  and 47.5% (56/118) of the samples had FC27 and 3D7 allelic types in the 2006–2007 group , FC27-6 (P < 0.01), FC27-7 (P < 0.01), FC27-10 (P < 0.01), FC27-11 (P < 0.01), FC27-16 (P < 0.01), and FC27-19 (P < 0.01), and 3D7-2 (P < 0.01), 3D7-4 (P < 0.01), 3D7-5 (P < 0.05), 3D7-13 (P < 0.01), and 3D7-15 (P < 0.05) were significantly decreased in the 2013–2016 group, whereas the frequencies of FC27-14, 3D7-7, 3D7-9, and 3D7-12 haplotypes were significantly increased (P < 0.01). Some of the P. falciparum Grande Comore isolates with msp-2 FC27 and 3D7 haplotypes in this study showed 100% identity with other strains from Vietnam (AAG47596 with FC27-20), while other haplotypes  were new alleles identified in this study.Fragments of the P. falciparum samples obtained from Grande Comore, 204 were successfully amplified and sequenced for msp-3 (105 for the 2006–2007 group and 99 for the 2013–2016 group). Sequence analysis of msp-3 showed that the allelic diversity of msp-3 allelic types (3D7 and K1) was attributed to variation in size of the AHR region, with K1 allelic type being 366 or 369 bp long and 3D7 type having 339 bp , whereas the frequencies of K1-2 and 3D7-1 haplotypes was significantly increased (P < 0.01).Of the 232 confirmed P. falciparum patients, ACT-based MDA, and other malaria control interventions. However, malaria continues to be one of most important public health problems on this island, which calls for monitoring changes in drug resistance status and parasite population dynamics. Determining P. falciparum genetic diversity and MOI from field samples is important for understanding the impacts of malaria control measures on parasite populations and for developing strategies to better control malaria infection. The present study investigates the temporal change of genetic diversity and MOI of Grande Comore P. falciparum populations based on msp-1, msp-2, and msp-3 genes that have been used to monitor parasite population widely [msp-1, msp-2, and msp-3 significantly decreased in the 2013–2016 group when compared with those the 2006–2007 group, which may reflect a trend associated with decreasing malaria transmission intensity on this island.Dramatic reduction in annual malaria cases has been achieved in Grande Comore through the use of ACT for the treatment of uncomplicated n widely , 15. Themsp-1 K1, MAD20, or RO33 haplotypes found in the present study have been reported in other regions of the world, including Brazil (AFS44739 with K1-5), Senegal , Malawi (ADQ74224 with K1-12 and ADQ74227 with RO33-1), India (AFR61077 with K1-12 and AFR61093 with MAD20-3), Tanzania (BAM84405 with K1-12 and AAC69748 with RO33-1), Colombia (ACS26173 with MAD20-6), and Myanmar (ACB69813 with MAD20-6); whereas the other allele variants of msp-1 were new alleles identified in this study. The present data reveal RO33 being the dominant allelic type of msp-1 gene among Grande Comore P. falciparum isolates in both groups. RO33 has also been reported to the dominant allele in parasites collected from Malaysia [msp-1 allelic type. For msp-2, the 3D7 type was the predominant among isolates in 2006–2007 group, similar to reports from several African countries, including Gambia [msp-2 gene is dramatically decreased, from 90.8 to 37.1%, whereas FC27 allelic type increased from 71.6 to 91.1%.Some Malaysia , and GabMalaysia , 19, ThaMalaysia , Iran [2Malaysia , and ColMalaysia , 24. ForMalaysia , Kenya [g Gambia , Congo [g Gambia , Ghana [g Gambia , 32, Seng Gambia , 23, 24,g Gambia . The 3D7g Gambia , Iran [2g Gambia , Myanmarg Gambia , Papua Ng Gambia , Thailang Gambia , as wellg Gambia . In contg Gambia . Severalmsp-1 and msp-2 gene, respectively, in 2006–2007 group. The frequencies of polyclonal infections were reduced to about 29 and 28%, respectively, in the 2013–2016 group, which again suggests decreasing population diversity and/or transmission intensity. MOI is conventional index to measure of complexity of infection and intensity of transmission. A high MOI value is often observed in a hyperendemic region with high malaria transmission [msp-1 and from 2.75 to 1.35 for msp-2, respectively. The findings in this study are similar to those reported in southeastern Senegal [P. falciparum transmission on this island. In fact, according to a report from the Comoros Ministry of Health, the numbers of annual malaria cases in Grande Comore dramatically decreased from 92,480 (in 2006) with high level incidence (about 35%) to 1362 (in 2016) with low level incidence (about 0.3%). Therefore, the present data confirm that MOI can be used as a useful indicator for monitoring malaria transmission level in the endemic areas.Previous reports show that polyclonal infection is more common in areas with high endemicity, and 50–100% of infections are polyclonal infections in mesoendemic and holoendemic areas –45. Furtsmission , 47, 48. Senegal  and Cong Senegal . The premsp-1 haplotypes (32 for the 2013–2016 and 23 for the 2006–2007 group) among Grande Comore isolates. Similarly, the total number of haplotypes in msp-2 dramatically decreased from 29 in the 2006–2007 group  to 21  in 2013–2016 group (about 28% decline). This is in agreement with previous reports in other countries with declining endemicity [msp-2 gene among P. falciparum isolates from children living in Southern districts of Brazzaville [Sequence analysis revealed that the decline in the number of demicity , 50. Howzzaville . Again, pfmsp-3 is predominantly confined to sequence diversity in the N-terminal domain within the heptad-repeats (insertion/deletion and nucleotide substitutions) [msp-3 K1 and 3D7, but not recombinant type, similar to those reported from Thailand, Papua New Guinea, India, Keyan [msp-3 alleles were detected in Iran and African countries at a very low frequency [msp-3 3D7-1 haplotype was the most prevalent in both 2006–2007 and 2013–2016 groups. The present data were in line with the findings from Thailand, India, and Nigeria, where 3D7-1 haplotypes was the most abundant types [P < 0.01), while the 3D7 type dramatically increased from 33.3 to 53.5% (P < 0.01), suggesting that parasites with the msp-3 3D7 type may survive better after introduction of ACTs in Grande Comore. The total number of haplotypes in msp-3 gene changed from 11 in 2006–2007 to 3 in 2013–2016 (a 60% decline), suggesting a decreasing tend in genetic diversity of msp-3 in Grande Comore after 10 years of use of ACT.The polymorphism of tutions) . The prea, Keyan . Recombirequency , 52. Somnt types . In the nt types , Thailannt types , and Thant types , but is nt types  and Perunt types , with thmsp-2 FC27 and msp-3 3D7 allelic types when general population genetic diversity and other allelic types have decreased are interesting, although we do not know the reason for the shift of the alleles. One remote possibility is that the msp-2 FC27 and/or msp-3 3D7 alleles or some unknown genes nearby (linked to msp-2 and/or msp-3) play a role in parasite response to ACT. Parasites carrying these specific alleles/genes can survive better under drug pressure and increase frequency. Another possibility is that the 2013–2016 parasite populations might consist of some parasites carrying these alleles imported from nearby endemic regions after reduction in the original parasite populations. These issues require further investigations.The observation of increased frequencies of P. falciparum populations in Grande Comore Island after the introduction of ACT using the polymorphic genetic markers . Results from the current study showed that the prevalence of genetic diversity and MOI in msp-1, msp-2, or msp-3 decreased over the course of the study (July 2006 to July 2016). The data in this study suggest a progressive decrease in genetic diversity likely due to lower malaria transmission intensity. The data presented here provide a valuable information for assessing the appropriateness of the current malarial control strategies in this endemic area.This study investigated the temporal change in genetic diversity and MOI of


Here we review the contribution of transcriptomic studies to the understanding of malaria, a parasitic disease which has exerted a major influence on human evolution and continues to cause a huge global burden of disease. We consider malaria a paradigm for the transcriptomic assessment of systemic host-pathogen interactions in humans, because much of the direct host-pathogen interaction occurs within the blood, a readily sampled compartment of the body. We illustrate lessons learned from transcriptomic studies of malaria and how these lessons may guide studies of host-pathogen interactions in other infectious diseases. We propose that the potential of transcriptomic studies to improve the understanding of malaria as a disease remains partly untapped because of limitations in study design rather than as a consequence of technological constraints. Further advances will require the integration of transcriptomic data with analytical approaches from other scientific disciplines, including epidemiology and mathematical modeling.Transcriptomics, the analysis of genome-wide RNA expression, is a common approach to investigate host and pathogen processes in infectious diseases. Technical and bioinformatic advances have permitted increasingly thorough analyses of the association of RNA expression with fundamental biology, immunity, pathogenesis, diagnosis, and prognosis. Transcriptomic approaches can now be used to realize a previously unattainable goal, the simultaneous study of RNA expression in host and pathogen, in order to better understand their interactions. This exciting prospect is not without challenges, especially as focus moves from interactions  Transcriptomics is the quantitative or qualitative study of RNAs on a genome-wide scale . It is jInitially, transcriptomic studies sought to quantify the expression levels of protein-encoding genes, often with the implicit assumption that this would broadly indicate changes in protein expression levels . However4–7–Plasmodium, which can infect a diverse range of vertebrate hosts. A comprehensive description of malaria epidemiology, biology, immunology, pathogenesis, and treatment is beyond the scope of this text and has been covered in recent review articles  quickly make their way from the skin to blood vessels to hepatocytes, where they undergo massive intracellular asexual replication during the incubation phase of infection  (–For (rigors) –13. OfteIf parasite numbers continue to increase, because of insufficient constraint by the host response or a failure to receive antimalaria treatment, this predisposes an individual to an increasing risk of severe disease manifestations, which may include coma, lung injury, renal failure, acidosis, and severe anemia , 13, 16.P. falciparum and is largely explained by specific adhesive interactions between vascular endothelial surface molecules and parasite molecules expressed on the surface of infected RBCs (iRBCs)  –19. Howe(iRBCs) –. The sex(iRBCs) –.Individuals in some areas where malaria is highly endemic will be exposed to multiple infectious mosquito bites every day, and cumulative infections result in the acquisition of clinical immunity. First, individuals cease to be vulnerable to severe disease, and they then become less likely to develop clinical symptoms . This clClinical manifestations of malaria are due to the asexual blood stage of the parasite, and the host-parasite interactions that cause disease occur within the vasculature and its contiguous organs, such as the spleen , 16, 22.26–The pathogen load is likely to be an important factor in determining the pathogenesis of many infectious diseases but is much harder to quantify as a stimulus for the systemic host response when pathogens are differentially distributed throughout multiple tissues . On a reHere we aim to describe the contribution that transcriptomic studies have already made to understanding malaria and highlight how new approaches might permit greater insights. We provide an introduction to existing and new technologies and analytical approaches and outline how these technologies are transforming the depth and richness of transcriptomic data. We then consider malaria as a paradigm for transcriptional analyses of systemic host-pathogen interactions, lessons learned from malaria studies, and strategies for application to other infectious diseases. We illustrate that the traditional approach of considering variation in the host response to an invariant pathogen is too simplistic and that accumulating evidence suggests that dynamic variation in pathogen behavior also needs to be considered.There are numerous excellent reviews of transcriptomic technologies, including comparisons of their relative merits and detailed methodological considerations , 33–37, 33–In theory, RNA-seq is much better suited for studies of host-pathogen interactions, although this is not always straightforward , 56. A min vivo, and deep RNA sequencing is already achieving some success   mapping to the parasite genome  . In conte genome , which l RNA-seq . TherefoPlasmodium species in vitro and in vivo. Here we focus primarily on those using samples from humans with P. falciparum malaria and animal models of human infection, and we draw upon pivotal in vitro studies, where necessary, to give context. Recent reviews have addressed the application of transcriptomics to specific aspects of malaria immunology, vaccinology, and host-parasite interactions , which can confound the interpretation of the transcriptome obtained from analysis of mixed stages. Several analytical approaches have been developed to try to estimate the developmental stage of parasites, including a maximum likelihood approach using global gene expression  can be misleading if no consideration is given to the composition of the mixture of parasite stages  variants, antigenically diverse proteins expressed on the surface of iRBCs  or binding to uninfected RBCs (a phenomenon known as rosetting), both of which are correlated with virulence  multigene family can be found in every Plasmodium genome that has been sequenced so far. This vast gene family includes large numbers of gene loci in each species, including stevor (∼40 loci) and rif (∼180 loci) in P. falciparum, ∼180 loci in P. berghei, ∼800 loci in P. yoelii, ∼200 loci in P. chabaudi, ∼68 loci in P. knowlesi, ∼1,200 loci in P. cynomolgi and P. vivax, ∼250 loci in P. malariae, and nearly 2,000 loci in P. ovale  genes is strongly influenced by whether mice are infected by a mosquito bite or serial blood passage, and these modes of transmission also determine parasite virulence  suggests that parasite gene expression is generally well conserved between laboratory-adapted P. falciparum strains and parasites directly sampled from naturally infected humans Plasmodiue stages , 21. Enoe stages , most ofTranscriptomic studies therefore play an important role in and are potential building blocks for an integrated description of immune responses to malaria. It would be ideal to obtain serial samples from all relevant tissues, starting before inoculation by a mosquito bite, through the presymptomatic and symptomatic phases of infection, and onwards until the time of death, resolution, or persistent infection. This may be possible in animal models , but human studies are often constrained by the limited availability of any sample type other than blood and the practical and ethical challenges of longitudinal sampling . Thus, wInsights into some of the earliest immune responses to blood-stage parasites in humans come from controlled-infection studies, whereby malaria-naive individuals are infected by a mosquito bite or the inoculation of blood-stage parasites and then intensively monitored for the detection of parasites in their blood , 123. AnGene expression profiles from naturally infected individuals at the time of clinical presentation with acute uncomplicated malaria (UM) show many similarities with those of presymptomatic experimentally induced infections , 124–126124–124–Differences between the transcriptional profiles seen in cases of uncomplicated and severe malaria have been less studied. A paired comparison of 5 individuals who first presented with severe malaria and later returned with an episode of uncomplicated malaria found that IFN pathway and T-cell response genes were more highly expressed in the uncomplicated episodes than in the severe episodes . AlthougPlasmodium infections, transcriptomic approaches have been used to understand the consequences that one or more episodes of malaria may have on subsequent responses to Plasmodium or other pathogens. Comparison of PBMC transcriptomes of Malian children 7 days after treatment for the first acute episode of the malaria season with transcriptomes just before the onset of the malaria season revealed the downregulation of inflammatory genes but the upregulation of genes expected to facilitate microbial killing and the activation of adaptive immunity  were examined at different time points during P. yoelii 17XNL-infected BALB/c mice to resolve a controversy surrounding their function in malaria -vectored CSP or two doses of DNA multiple-epitope thrombospondin-related adhesive protein (ME-TRAP) followed by MVA-vectored ME-TRAP, showed that the small number of protected subjects were characterized by the upregulation of interferon-induced and antigen presentation genes and the downregulation of hematopoietic stem cell and myeloid cell genes  enables immune evasion and is at least partly determined by immune selection. For the Pc-PIR genes of P. chabaudi, this appears not to be the case. Transcriptomic comparisons of parasite gene expression levels in mice with acute and chronic infections demonstrated that the establishment of chronic infection was indeed associated with a clear shift in Pc-PIR gene expression, but this was not influenced by the removal of immune selection in mice without T cells (TCRα−/− mice) or without B cells and antibodies (μMT mice)  alters parasite behavior. Transcriptomic analyses, particularly dual RNA-seq of host and parasite, could provide insights into this question. However, some surprising findings have arisen from studying the more fundamental question of whether the presence or absence of specific components of host immunity alters parasite gene expression. A common assumption has been that the expression of different members of parasite variable gene families (such as MT mice) . This fiimmunity .Malaria typically causes fevers, headache, myalgia, rigors, cough, and abdominal pain, features which are similar to those of many other systemic infections , 12. Lab169–P. berghei ANKA strain causes a neurological syndrome described as ECM  has been extensively debated because it is currently impossible to prove the dependency of the syndrome on any specific pathogenic mechanism. In contrast to the widely used subtypes –184. Theubtypes –. In resoubtypes –, 185. Thubtypes –, 186.Few studies have compared the human or mouse blood transcriptomes between CM and uncomplicated malaria , 171 or P. berghei ANKA P. berghehei ANKA . In conthei ANKA . Howeverhei ANKA . A diffehei ANKA .In contrast to the limited studies on blood, multiple transcriptomic analyses of mouse brain tissue have been conducted and have yielded a fairly consistent picture of gene expression associated with ECM , 177–179177–174–174–In some comparisons, an upregulation of type I interferon responses was found to precede the onset of cerebral pathology , 144, whBeyond implicating immunopathological mechanisms initiating ECM, brain transcriptomes have also revealed possible explanations for neurological dysfunction. The increased expression and activation of apoptosis pathways were observed in several studies, along with a variety of cellular stress response pathways –178. How–+ dendritic cells  , 138. Spic cells , which mble mice  but was ble mice .In an attempt to achieve a more integrated understanding, sequential changes in gene expression in spleen, brain, lung, and liver were examined in comparisons between ECM-susceptible and -resistant mice . Large gvar genes and severity but beyond this have been rather inconclusive. Targeted analysis of the var transcriptome revealed that severe malaria (both CM and severe anemia) was associated with high expression levels of var genes encoding PfEMP1 variants with cysteine-rich interdomain regions predicted to bind to the endothelial protein C receptor , young rats failed to control parasitemia and died, while older rats controlled parasitemia, dependent on the differential expression of a small set of genes in the spleen, mostly related to T-cell function  and immunohistochemical analyses, led the authors of that study to speculate that macrophage CXCL13 drives B-cell recruitment to the placenta, antibody production, and further antibody-mediated activation of inflammation, in a pattern suggestive of lymphoid neogenesis  . Howeverin vitro . That sain vitro .Comparison of parasite gene expression levels has been performed with a small number of adults with noncerebral severe malaria  versus those with uncomplicated malaria by using a custom microarray designed to identify a broader repertoire of genes from variant gene families of parasites . Given tvar2csa, many of which are thought to be exported into erythrocytes but do not have well-defined functions  were different enough between groups to be considered candidate biomarkers. C1Q was also identified as a putative biomarker of severe human malaria in a pilot study of 10 Malian children Plasmodiuerferons , whole berferons , 173, anerferons .In contrast, increases in type I interferon-related gene expression levels in brain are temporally associated with the onset of ECM , 179, whNeutrophils are one of the most numerous leukocyte populations in blood, yet there has been disproportionately little investigation of their role in malaria. Higher neutrophil-associated gene expression levels are a consistent feature of analyses of whole-blood transcriptomes from uncomplicated and severe human disease , 187. InRelative neutrophilia is frequently described in human cases of malaria , 222, paConsistent with this, immature neutrophils and elevated circulating levels of their granule proteins are detected in cases of severe and uncomplicated malaria , 224. Ma226–Proving a functional role for neutrophils in rodent model pathogenesis, and extrapolating the data to human disease, is also challenging. Antibody depletion of neutrophils has been used to assess their role, but common antibodies used for this purpose can also deplete monocyte subpopulations , 230. Dein vivo and in vitro parasite transcriptomes, particularly after accounting for the parasite developmental stage, is discussed above. However, the amount of variation from the in vitro “standard” that occurs in vivo remains to be fully quantified, as do the implications of any in vivo variation. Differences in parasite gene expression detected between different in vivo situations  may reveal gene expression common to all severe phenotypes and gene expression specific to each one. Since the severe phenotypes share many common risk factors, such as parasite load , we woulEven stronger evidence may come from one of the most powerful epidemiological methods for demonstrating causation, Mendelian randomization . This epin vitro or in animal models for experimental validation. In humans, causality can be assessed directly in interventional studies, with transcriptomics providing a means to elucidate the mechanism of action of new treatments in clinical trials.Regardless of the approach, new hypotheses may be generated based on gene expression data, which can be tested by using reductionist approaches It is clear that many studies on malaria had suboptimal elements in their design. Principles for improving the design of malaria studies will also apply to many other infectious diseases . The oveFifteen years of transcriptomic studies of malaria have brought many insights but also highlighted the complexity and challenges of studying host-pathogen interactions in this way. For other infectious diseases, some of these challenges may be even greater. In the next decade, advances in transcriptomics will likely transform our understanding of host-pathogen interactions in many infections. Specific examples might include unraveling the triggers and consequences of bacterial toxin and protease production in virulent staphylococcal and streptococcal infections; establishing the mechanisms underlying clearance, latency, or progression in tuberculosis; and defining protective and harmful host-pathogen interactions in emerging infections, such as Ebola and Zika viruses.This will be facilitated by technical advances, such as long-read sequencing technologies and single-cell sequencing, that will allow unprecedented levels of detail to be described. The greatest challenge may become the synthesis of the huge amounts of data and detail into a model that is comprehensible to the human mind.We believe that important insights will come from the integration of multiple layers of data, such as transcriptomic, genomic, proteomic, and metabolomic data , combineTo ensure the translation of discovery to clinical application, researchers will need to better characterize the clinically relevant components of their model systems and achieve optimal sampling of tissues with the most-relevant host-pathogen interactions. This will require collaboration across disciplines, bringing transcriptomics together with advances in imaging, minimally invasive sampling, microscopy, and viable-cell separation. Studies with humans will be greatly enhanced by embedding within formal epidemiological studies with rigorous design principles. Clinical trials of antimicrobials and adjunctive therapies represent rare opportunities where humans are subjected to experimental manipulation, and collecting samples for transcriptomic analyses will be helpful for mechanistic interpretations of their results.Mathematical developments will likely facilitate new approaches to studying trajectories of infectious diseases, overcoming the inherent limitation that naturally infected human subjects present only a snapshot view of the dynamic process of infection . ApproacTranscriptomic studies fairly consistently show that different gene expression signatures characterize infections with different pathogens and different stages of infection with the same pathogens , 211. ThIn conclusion, we are optimistic about the advances in the understanding of host-pathogen interactions that will come from future transcriptomic studies and the benefits that may come from these studies if they are well conducted. We predict that the results of these studies will force researchers to consider much more dynamic models of infection, where changes in the host response may affect pathogen behavior and vice versa, and that the simultaneous study of host and pathogen will increasingly be viewed as being essential.


Plasmodium falciparum and non-P. falciparum status of symptomatic and non-symptomatic residents of Mpraeso in the highlands of Kwahu-South district of Ghana based on evidence of current national decline. The influence of HIV and SCD on malaria was also determined.In the past two decades, there has been a reported decline in malaria in Ghana and the rest of the world; yet it remains the number one cause of mortality and morbidity. Human immuno-deficiency virus (HIV) and sickle cell disease (SCD) share a common geographical space with malaria in sub-Saharan Africa and an interaction between these three conditions has been suggested. This study determined the Participants were 354 symptomatic patients visiting the Kwahu Government Hospital and 360 asymptomatic residents of the district capital. This cross-sectional study was conducted during the minor rainy season (October–December 2014). Rapid diagnostic tests (RDT), blood film microscopy and real-time polymerase chain reaction assessment of blood were done. Participants who tested positive with RDT were treated with artemisinin-based combination therapy; and assessment of venous blood was repeated 7 days after treatment. HIV screening and haemoglobin genotyping was done. Univariate and multivariate regression analysis was used to determine the influence of SCD and HIV.Plasmodium falciparum was prevalent at 124/142 (87.3%). Plasmodium malariae was the only non-falciparum species detected at 18/142 (12.7%). HIV and SCD did not significantly increase odds of malaria infection. However, the use of ITN and recent anti-malarial intake significantly decreased the odds of being malaria infected by 0.45-fold and 0.46-fold respectively.Plasmodium falciparum and P. malariae infection are the prevailing species in the study area; albeit varying from the national average. HIV and SCD were not associated with the risk of having malaria.The online version of this article (doi:10.1186/s12936-017-1823-y) contains supplementary material, which is available to authorized users. Current indications are that malaria is on the decline in Ghana and other countries –5. This Plasmodium species currently known to infect humans—Plasmodium falciparum, Plasmodium ovale, Plasmodium malariae, Plasmodium vivax and Plasmodium knowlesi—only the first three are documented to occur in Ghana [Plasmodium falciparum is the prevailing species (90–98%), with few P. malariae (2–9%) and P. ovale (1%) [P. ovale wallikeri (variant type) and P. ovale curtisi (classic type), which have been identified across sub-Saharan Africa [This significant decrease in prevalence of malaria in Ghana may result in marked changes in the current species dominance. Of the five in Ghana . Plasmodale (1%) , infectin Africa –8 and As species 0–98%, win Africa .P. malariae [Plasmodium spp. The aim of this study was, therefore, to evaluate the state of P. falciparum and non-P. falciparum infections in a highland in the Eastern region of Ghana; and to determine the possible influence of SCD and HIV.The malaria hypothesis has shown that sickle cell disease (SCD) and malaria share a common geographical space . Ghana, malariae . Howevermalariae , 18. TheGhana is divided into three malaria epidemiological zones; namely, northern savannah, tropical rainforest and coastal savannah and mangrove swamps . The stuThe first group of study participants was recruited from the Kwahu Government Hospital, which is one of two district hospitals that serve communities in the entire Kwahu mountain ridge and beyond . An estiP. falciparum infections are artesunate–amodiaquine (AS+AQ), artemether–lumefantrine (A–L) and dihydroartemisinin–piperaquine (DHAP); the second line drug is oral quinine (QN) for 7 days [In Ghana, anyone presenting at the health facililty with axillary temperature ≥37.5 °C without any other symptom is suspected of malaria . The curr 7 days , 24. IntThis was a cross-sectional study, done during the minor rainy season, from October to December in 2014. Patients presenting at the OPD with an axillary temperature ≥37.5 °C with or without vomiting, nausea, general malaise, headaches or body aches were recruited into this study and classified as symptomatic. There was no age or sex discrimination in participant selection. Demographic data and medical history of participants were collected by trained collectors prior to sample collection, and after obtaining written or finger-stamped informed consent. Demographic data included age, sex and education whilst medical history included axillary temperature, symptoms, diabetes status and recent anti-malarials taken. In instances where participants could neither read nor write, questions were asked in the local language and their responses were filled in by the trained sample collectors. For children under the age of 18 years, their parents/guardians provided responses on their behalf.® Malaria Ag. Plasmodium falciparum (HRP2), to test for the presence of P. falciparum. Patients who tested positive for malaria, with either RDT or microscopy, were prescribed the first-line artemisinin-based combination therapy (ACT) artemether–lumefantrine by the attending physician, according to the national malaria control programme. It is a fixed-dose formulation of 20 mg artemether and 120 mg lumefantrine per tablet [Expert phlebotomists took venous blood from the antecubital fossa of participating patients at the hospital. This was stored in ethylenediaminetetraacetic acid (EDTA) tubes at 4 °C until processed. Participants from the community had their finger-prick capillary blood taken. Thick and thin blood smears were prepared from all samples. After air-drying, thin smears were fixed in 100% methanol. All smears were stained with 10% Giemsa which was applied to both thick and thin films for 10 min. These were then examined by two expert microscopists. Samples were recorded as positive under 200 high powered visual fields if any amount of asexual parasites was observed per 200 white blood cells, and negative if none was observed. Discordant results were decided on by a third microscopist. A drop of each venous blood sample taken was applied to the malaria RDT First Responser tablet . This isr tablet . Patientr tablet  was donePlasmodium. Species identification was done, on positive samples, with nested PCR amplification of small sub-unit ribosomal RNA genes as previously described [P. ovale wallikeri and P. ovale curtisi as described [Two to three drops of each blood sample was spotted onto Whatman filter paper (grade 3), dried and stored and PCR analysis done at KIT Biomedical Research Institute in Amsterdam. Boom extraction, as previously described , was useescribed . Gel eleescribed .Blood samples of hospital participants were further tested for HIV using INSTANTCHEK HIV-1 + 2 rapid diagnostic test kit by EY Laboratories and Oraquick Advance Rapid HIV-1/2 antibody test by OraSure Technologies, Inc. The use of Oraquick Advance Rapid HIV-1/2 antibody test is consistent with the confirmatory testing protocol for HIV in Ghana .P value ≤0.05 was regarded as statistically significant.Data was entered into Microsoft Excel and then transferred into IBM SPSS version 20 software  for statistical analyses. Univariate and multivariate regression analysis were used in determining whether primary outcome of malaria positivity was influenced by several independent variables, after adjusting for confounders. Independent variables included sex, sickle cell status, HIV, diabetic status and anti-malarial history. The strength of association was measured by odds-ratio with a confidence interval of 95%. The baseline medical and demographic history of participants from the OPD and community is summarized in Table P. malariae was present  was found in 86/124 (69.4%) participants with falciparum malaria; and in 11/18 (61.1%) of participants with non-falciparum malaria. In both symptomatics and asymptomatics, malaria was prevalent in participants who had not taken anti-malarials recently ; the use of ITN showed a significant 0.45-fold decrease in odds for malaria. Data and anyalysis for this study may be found in supplementary file  whilst non-falciparum infections were 18/142 (12.7%)  in different parts of the country [P. vivax was not detected in this area. HIV and SCD did not influence malaria in this particular study.Malaria prevalence and determinants as well as prevailing species in this highland of Ghana varied from the published prevalence of malaria in that administrative region of Ghana. This might be because of the heterogeneous nature of malaria transmission in the country. The prevalence in this study was 3.2% lower than prevalence published in the national survey . Only twin Ghana , and per country , was absP. ovale curtisi or P. ovale wallikeri infections in this study. Both P. ovale curtisi and P. ovale wallikeri have been found to circulate in the population in Ashanti region [P. ovale wallikeri was diagnosed in a foreign visitor to the country who visited different regions in the country [P. malariae in this study was much higher at 12.7% compared to that of the national prevalence of 2–9% [Unlike studies done elsewhere in the country , 32, thei region ; whilst  country , P. mala of 2–9% . These d of 2–9% , 34. Mal of 2–9% . In thisPCR testing of malaria positive patients 7 days post treatment indicated parasite clearance in 80 out of 93 samples. A previous study in that area showed residents combined anti-malarials with local herbal preparations when treating malaria , and thiUnivariate and multivariate regression analysis of the risk of having malaria in symptomatic participants showed that HIV status and sickling status did not cause any significant increase or decrease in risk, even though 14.4 and 17.7% of participants carried the HbS allele and had HIV respectively. In this study neither HbSS nor HbAC showed any influence on malaria; other studies have also been unable to show a clear relationship between them , 18, 36.Despite the Ghana national policy on intermittent preventive treatment in pregnancy (IPTp), which advocates the administration of sulfadoxine-pyrimethamine at monthly intervals after 16 weeks , 22.3% oNonetheless malaria and malaria-related deaths have declined in the past 6 years , 44–47. Plasmodium species prevalence and HIV status of all participants. Participants in the hospitals were more willing to have their HIV status determined; no one in the community aspect of the study agreed to the test. Thus, the absence of statistical findings in this study may not necessarily mean the absence of parasites or correlations, and larger cohort studies are therefore needed. Secondly, even though majority of patients returned for the first follow-up, none returned for the second. This prevented information on subsequent parasite clearance from being obtained. Thirdly, this study was done during the minor rainy season. An extension of the study to include both major and minor rainy seasons would have provided a clearer interpretation of the influence of rainy seasons on malaria.This study had certain limitations. Firstly, the study sample was small and therefore may not have been adequately sized to accurately measure local Plasmodium spp. type and prevalence in this study area varies from the other regions in Ghana. The high prevalence of the HbS allele in the study population did not significantly influence malaria; neither did HIV. Nonetheless, further large-scale studies are necessary to identify possible influence of SCD and HIV as has been suggested to occur elsewhere.


Malaria is a major public health problem and an important cause of maternal and infant morbidity in sub-Saharan Africa, including Ethiopia. Early and accurate diagnosis of malaria with effective treatment is the best strategy for prevention and control of complications during pregnancy and infant morbidity and mortality. However, laboratory diagnosis has relied on the identification of malaria parasites and parasite antigens in peripheral blood using Giemsa-stained microscopy or rapid diagnostic tests (RDTs) which lack analytical and clinical sensitivity. The aim of this study was to evaluate the performance of loop-mediated isothermal amplification (LAMP) for the diagnosis of malaria among malaria suspected pregnant women in Northwest Ethiopia.Plasmodium parasites by microscopy, RDT, and LAMP. Diagnostic accuracy outcome measures  of microscopy, RDT and LAMP were compared to nested polymerase chain reaction (nPCR) as the gold standard. Specimen processing and reporting times were documented.A cross sectional study was conducted from January to April 2016. Pregnant women (n = 87) suspected of having malaria at six health centres were enrolled. A venous blood sample was collected from each study subject, and analysed for Using nPCR as the gold standard technique, the sensitivity of microscopy and RDT was 90 and 70%, and the specificity was 98.7 and 97.4%, respectively. LAMP assay was 100% sensitive and 93.5% specific compared to nPCR.This study showed higher sensitivity of LAMP compared to microscopy and RDT for the detection of malaria in pregnancy. Increased sensitivity and ease of use with LAMP in point-of-care testing for malaria in pregnancy was noted. LAMP warrants further evaluation in intermittent screening and treatment programmes in pregnancy. Plasmodium falciparum is the principal cause of severe malaria while Plasmodium vivax is increasingly recognized as capable of causing severe disease [ disease –4. Accor disease .P. falciparum, in pregnant women increases the risk of maternal death, miscarriage, stillbirth and neonatal death [Every year about 30 million African women in malaria-endemic areas become pregnant and are at risk of infection with malaria, and an estimated 75,000–200,000 infant deaths are reported due to malaria infection in pregnancy . Malariaal death . The impal death , 10.P. falciparum resistance to SP in sub-Saharan regions, the use of rapid diagnostic tests to screen women for malaria at the first or each antenatal visit and then treat is likely more sustainable than IPTp without diagnosis [The World Health Organization (WHO) recommends a three-pronged approach to reduce the burden of malaria in pregnancy: (1) provision and promotion of insecticide treated bet nets (ITN) or long-lasting insecticide- treated bed nets (LLINS); (2) administration of intermittent preventive treatment with sulfadoxine–pyrimethamine (IPTp-SP) after the first trimester of pregnancy in areas with stable malaria transmission; and (3) prompt diagnosis and appropriate treatment of malaria , 11. Howiagnosis .Early and accurate diagnosis with effective treatment is the best strategy to decrease malaria-related pregnancy complications and infant mortality. The current malaria diagnostic methods include Giemsa-based microscopy, rapid diagnostic tests (RDTs), polymerase chain reaction (PCR) and placental histology depending on the setting . The pooLoop-mediated isothermal amplification (LAMP) is a simpler molecular procedure and better alternative for the field than nPCR , 19. LAMPlasmodium vivax and year-long P. falciparum exist in the area [The study was conducted at Kola Diba, Girargie, Aymba, Azezo, Tseda, and Sanja Health Centres, which are located in North Gondar, Amhara Regional State, Ethiopia. The district covers an area of 1270 km with a total population of about 263,000 people approximately 700 km north of Addis Ababa close to Lake Tana. The ethnic group which predominates in this region are the Amhara people. The district sits at an altitude between 1750 and 2100 m above sea level. Malaria is the most prevalent seasonal disease in the area, and is the second most common reportable disease at the health centre. Both seasonal the area . Peak maA cross-sectional study was conducted among malaria suspected pregnant women at six health centres in Northwest Ethiopia from January to April , 2016. Study participants were recruited consecutively (convenience sampling) and a total of 87 pregnant women with signs and symptoms consistent with malaria were enrolled. Pregnant women who provided the required laboratory sample for the study were included in the study. Those subjects who were severely ill according to WHO criteria and/or had received anti-malarial drugs during the past 4 weeks prior to study commencement were excluded.P. falciparum and lactate dehydrogenase for the diagnosis of non-falciparum species.Demographic profiles and clinical data were collected at the health centre antenatal clinic using an interview-based questionnaire translated into the vernacular language that the participant could understand. In addition, two milliliters of venous blood was collected from each study participant using an EDTA anti-coagulated test tube. Soon after venous blood collection, a drop of blood was taken for the RDT, then two separate drops of blood were placed on a frosted microscopic glass slide to prepare both thin and thick blood films, and another two separate drops of blood was placed on Whatman filter for nPCR. The rest of the blood was kept at 2–8 °C at the health centre then transported to the Medical Parasitology Laboratory, University of Gondar on weekly basis to be kept frozen until analysed with LAMP. Blood films were processed, and parasite detection and parasitaemia level was determined according to WHO standards . CareStaPlasmodium/P. falciparum DNA. The preparation of the LAMP mixture, sample running and detection was performed as described previously [g for 3 min. The supernatant (30 ml) was pipetted into a dilution tube containing 345 μl of sterile water, and finally 30 μl of diluted DNA sample was used in the LAMP assay. Reaction tubes containing extracted DNA sample were incubated at 65 °C in a water bath for 40 min. Amplification was detected by naked eye based on turbidity. Two additional blinded laboratory technicians read the turbidity observed at the end of the LAMP reaction for the final consensus result.Loopamp™ malaria Pan/Pf detection kits  which detect mitochondrial DNA were used to amplify eviously . BrieflyGenomic DNA was extracted from dried blood on Whatman filter paper 903  and nPCR analysis was performed as described previously .Data were entered into Microsoft Excel program and then analysed with SPSS version 20. Sensitivity, specificity, predictive values and Cohen’s kappa coefficient were determined using SISA online statistical software . All dis+5.2) years were enrolled into the study, of which 50.6% (45/87) had a previous malaria history ; three were P. vivax , and two were mixed infections of P. falciparum and P. vivax (parasitaemia density minimum/maximum: 520/5360 per μl). LAMP identified 15 positive (17.2%) specimens and RDT 9 (10.3%) positives. A higher positivity rate of both single and mixed Plasmodium species infection were reported by LAMP than other diagnostic techniques used in this study  age of 27.43  compared to microscopy  and RDT . Microscopy had the greatest specificity  compared to RDT  and LAMP . There were five discordant results between nPCR and LAMP where five of them were negative by nPCR but positive by LAMP. There was also one study participant with a parasitaemia load of 520/μl negative only by nPCR. In this study, LAMP showed better sensitivity but lower specificity than microscopy and RDTs using nPCR as the gold standard  of malaria infected pregnant women were multigravida. In contrast, a study from Gabon revealed that primigravida and young pregnant women were associated with increased malaria susceptibility. Although not statistically significant, based on microscopy results, primigravida women demonstrated a higher median parasite density (5200 parasites/μl) than multigravida (1560 parasites/μl) . This isPolymerase chain reaction (PCR) in its various formats has emerged as the most sensitive method able to detect low levels of parasites in the blood especially in this setting , 37. Howbona fide gold standard could be questioned in this study as it relied on filter paper samples which may be subject to DNA degradation in transport to Canada from Ethiopia. DNA degradation on filter paper may ultimately explain the lower kappa value between nPCR and LAMP versus nPCR and microscopy. In support of this supposition, one specimen was positive by all three methods except nPCR. Thus, additional LAMP positive cases may be true positives and warrant treatment. However, others have reported that genomic DNA is stable on filter papers over time making this less likely a contributing factor to the discordance observed [The current study showed lower specificity of LAMP (93.5%) than RDTs and Giemsa microscopy. This result was in line with a study conducted in Bangladesh where LAMP revealed lower specificity (58.3%) than microscopy and RDT (both 100%) when compared with nPCR . In the observed . All disobserved . To limiP. falciparum only by RDTs. Histidine rich protein-2 (HRP-2) antigen could persist for a long time even after effective treatment, giving false positive RDT test results in the absence of active P. falciparum infection [In the current study, two samples which were negative by RDTs ended up positive by LAMP, Giemsa microscopy and nPCR. For accurate diagnosis of malaria with RDTs, at least 100–500 parasites/μl of whole blood are required in peripheral blood. This was supported by a study from Tanzania which revealed that lower density of malaria parasitaemia is highly associated with the negative result of RDTs . In the nfection .Plasmodium parasites in intermittent screening programmes during pregnancy.Pregnant women are more vulnerable to malaria, harbour low level infections, and suffer malaria related complications themselves and to the neonate. It is important to use a highly sensitive, field-friendly detection method . LAMP wi