PfHRP) which is exclusively found in Plasmodium falciparum and a metabolic enzyme Plasmodium lactate dehydrogenase (pLDH) which is common among all Plasmodium species. Other RDTs incorporate the enzyme aldolase that is produced by all Plasmodium species. Recently, studies have reported false-negative RDTs primarily due to the deletion of the histidine-rich protein (pfhrp2 and pfhrp3) genes in field isolates of P. falciparum. Herein, we review published literature to establish pfhrp2/pfhrp3 deletions, the extent of these deletions in different geographical regions, and the implication in malaria control. We searched for publications on pfhrp2/pfhrp3 deletions and retrieved all publications that reported on this subject. Overall, 20 publications reported on pfhrp2/pfhrp3 deletions, and most of these studies were done in Central and South America, with very few in Asia and Africa. The few studies in Africa that reported on the occurrence of pfhrp2/pfhrp3 deletions rarely evaluated deletions on the flanking genes. More studies are required to evaluate the existence and extent of these gene deletions, whose presence may lead to delayed or missed treatment. This information will guide appropriate diagnostic approaches in the respective areas.Malaria remains the biggest threat to public health, especially among pregnant women and young children in sub-Saharan Africa. Prompt and accurate diagnosis is critical for effective case management and detection of drug resistance. Conventionally, microscopy and rapid diagnostic tests (RDTs) are the tools of choice for malaria diagnosis. RDTs are simple to use and have been extensively used in the diagnosis of malaria among travelers to malaria-endemic regions, routine case management, and surveillance studies. Most RDTs target the histidine-rich protein ( The previous two decade was characterized by remarkable progress in the fight against malaria, with more than seven million lives having been saved since 2001 . In 2015Plasmodium parasites, which is usually done by the use of microscopy or rapid diagnostic tests (RDTs) that target antigens/enzymes of the parasite in the patient’s blood. Although molecular techniques such as Polymerase Chain Reaction (PCR) provide high sensitivity and specificity for malaria diagnosis, the high cost, limited availability, and required expertise make them unsuitable as a diagnostic tool, especially in resource-limited settings [Diagnosis of malaria requires the identification of settings ,6.Plasmodium, which requires expert microscopists to differentiate them, deletions of the antigens targeted by RDTs, transmission intensity, parasite density, immunity, and the manifestation of clinical symptoms that are similar to other tropical diseases [Recently, the use of non-invasive specimens such as saliva and urine has been proposed as an alternative to blood samples. However, the utility of these approaches has not been validated ,8. Microdiseases ,10. Routdiseases .Plasmodium parasites in slides stained with Giemsa or Field’s stain [Plasmodium have shown that mixed species are common and are rarely detected by expert microscopists [Since Laveran discovered the malaria parasite and Romanowsky’s improvements on the staining techniques in the late 1800s, malaria has conventionally been diagnosed by the observation of ’s stain . Microsc’s stain . Malaria’s stain . However’s stain ,14,15. Mscopists . In rurascopists , and theTo overcome the apparent limitations of microscopy, the World Health Organization (WHO) has emphasized the urgent need for new, simple, quick, accurate, and cost-effective diagnostic tools to complement malaria microscopy . The advCurrently, there are about 60 manufacturers and 200 types of RDTs that have been tested and deployed in field settings . All RDTPlasmodium antigens that are detected in the same RDT. The histidine-rich proteins 2 and 3 (PfHRP2/3) are specific for P. falciparum, while Plasmodium lactate dehydrogenase (pLDH) and Plasmodium aldolase are made by all malaria species, hence the name pan-pLDH and pan-aldolase. Thus, in general, RDTs are sold as combinations of pan-pLDH-PfHRP2/3 or pan-aldolase-PfHRP2/3 [Malaria RDTs target three PfHRP2/3 .pLDH is a terminal enzyme in the glycolytic pathway of both the sexual and asexual stages of the parasite, and each of the four Plasmodium species has its specific isomer of this enzyme, while aldolase is conserved in all the species and is an example of a pan-malarial antigen target [pLDH catalyzes  the conversion of pyruvate into lactate and NAD+, (oxidised form of NAD) which is important for parasite survival in red blood cells [pLDH and aldolase are only produced in living Plasmodium cells and have a short half-life (2–4 days), the presence of these enzymes is an indication of current infection [pLDH either detect a pan-pLDH which is common to all human infecting species or specific regions unique to P. falciparum or P. vivax-specific pLDH [pLDH-detecting RDTs at extreme temperatures [P. falciparum [The n target . pLDH caod cells ,22. Sincnfection . RDTs thfic pLDH . Althougeratures , a studylciparum .pfhrp1 gene is associated with the expression of knob-like protrusions on the surface membrane of infected erythrocytes [Three histidine-rich proteins (HRPs) have been described, namely, HRP1 or the knob-associated histidine-rich protein (KAHRP), HRP2, and HRP3, also known as small histidine–alanine-rich proteins (SHARP) containing 8%, 35%, and 30% histidine repeats, respectively ,28. The hrocytes . These khrocytes .PfHRP2 is a water-soluble protein produced by the asexual stages and young gametocytes of P. falciparum and can be detected in serum, plasma, cerebrospinal fluid, and urine of infected patients and in the medium of in vitro cultured parasites [PfHRP2 have been shown to begin with immature rings and increase gradually as the asexual parasites mature such that, more than 90% is secreted during schizogony [PfHRP1, which is found only in knobby (K+) variants of P. falciparum, PfHRP2 is found in both the knobby and knobless (K-) strain variants [arasites ,33,34. Shizogony ,36, and hizogony . Unlike variants .pfhrp3 gene codes for a homologous protein histidine-rich protein-3 (PfHRP3). Both PfHRP2 and PfHRP3 are very similar in structure, although PfHRP3 has less histidine content [PfHRP2 with those of PfHRP3. Unlike PfHRP1 and PfHRP3, PfHRP2 is abundantly produced and is continuously expressed throughout the parasite life cycle.The  content . The strPfHRP2-based RDTs for malaria diagnosis has emerging limitations due to the existence of parasites with pfhrp2 gene deletions which, depending on the extent of the deletions, has led to false-negative results [pfhrp2 and pfhrp3 genes in laboratory lines [pfhrp2/pfhrp3 genes and/or the flanking genes [pfhrp2 and pfhrp3 genes, respectively   were folP. falciparum parasites lacking pfhrp2 and/ or pfhrp3 deletions was first reported [Published scientific articles were retrieved from PUBMED, MEDLINE, and Science Direct. We searched the scientific literature for articles published in English from January 2010 when the first case of reported , to DecePfHRP2, PfHRP3, pfhrp2/pfhrp3 gene deletions, and PfHRP2-RDTs. To filter out articles that were out of scope, we screened only for publications that were reporting on pfhrp2/pfhrp3 deletions. To be included in the analysis plan, a study had to fulfill stipulations as previously suggested [pfhrp2 and/or pfhrp3 genes. This is crucial to allow differentiation between lack of amplification due to gene deletions versus no amplification due to PCR failure.Keywords used for the search included: uggested , i.e., hData were entered in a Microsoft Excel database . The meta-analysis was conducted using the random effects model of analysis since it minimizes heterogeneity of the included studies. All of the analyses were implemented using R-3.6.2 metaprop and forest packages in order to estimate the pulled proportion, to investigate publication and other bias and to summarize information on individual studies, respectively. Descriptive statistics, such as bar charts, were used to summarize the distribution of reported deletions using the Statistical Package for Social Sciences (SPSS), version 25.pfhrp2 and pfhrp3 deletions. The final number of 20 publications that were reviewed is summarized in pfhrp2 and pfhrp3 genes in Peru and demonstrated that these parasites were widespread across the Peruvian Amazon [pfhrp2 (41%), pfhrp3 (70%) or both (21.6%) genes. The loss of these genes was confirmed by inability to detect the proteins in an immunological assay [pfhrp2 over a seven year period [Gamboa et al. (2010) reported the first evidence of field isolates lacking n Amazon . A huge al assay . A similr period .Pfhrp2 and pfhrp3 gene deletions have also been reported in P. falciparum isolates from Colombia within the Amazon River region, like in Peru [pfhrp2 deletions was lower than that of pfhrp3 . This is not surprising considering that the majority of parasites with pfhrp2 deletions were collected at the Peru border, where pfhrp2 deletions had been reported before. A study that evaluated the population structure of Colombian P. falciparum at the Colombian pacific coast revealed four different subpopulations  [pfhrp2 deleted parasites and 61.5% of the pfhrp2/ pfhrp3 double negatives were found to segregate in Cluster 2, which consisted of samples mostly collected from the Amazonas department that borders Peru and Colombia within the Amazon ecological zone, indicating expansion and a common genetic origin of pfhrp2 and pfhrp3 deleted parasites [ in Peru ,52, wherd Col-4) . Similararasites .pfhrp3 and not pfhrp2 deletions, recent reports have documented extensive deletions of both genes [pfhrp2 and pfhrp3 in South and Central Americas has increased over time, and there is a need to evaluate the selective pressures that favor the survival of parasites that lack these genes [Although earlier studies in Honduras only reported th genes ,54. The se genes .pfhrp2 and pfhrp3 and their corresponding flanking regions [pfhrp2, pfhrp3 and the flanking gene deletions in different continents. As shown in pfhrp2/pfhrp3 and the respective flanking gene deletions was reported in Asia. In one of the studies, deletions of the two genes were observed in symptomatic individuals with high parasitemia [pfhrp2 and pfhrp3 deletions in asymptomatic individuals with low parasitemia [Studies in India reported similar frequencies of parasites lacking  regions ,56,57. Fasitemia .pfhrp2/3 deletions, although a few surveillance studies have reported on their existence [In Africa, where malaria is endemic and the use of RDTs is widespread, there is scarce information on xistence ,63,68,69pfhrp2 gene deleted parasites were reported in Mali, where deletions were identified in parasitemic individuals whose blood was unreactive to PfHRP2-based RDTs [PfHRP2-based RDTs, Wurtz et al. (2013) did not find any difference in parasitemia between groups with pfhrp/pfhrp3 deletions and those without deletions, suggesting that, in addition to parasite density, lack of expression of the PfHRP2 may greatly influence the performance of RDTs. A recent study in Eritrea reported the emergence of false-negative PfHRP2-based RDTs that was not related to poor quality of RDTs, storage, handling or operator errors [pfhrp2 (62%) and pfhrp3 (82%), and that these parasites may have emerged due to local selective pressure since the parasite clusters obtained were not related to South American strains [The first cases of sed RDTs . In thatr errors . A subse strains .pfhrp2 deleted parasites in samples collected from asymptomatic children [pfhrp2 deleted samples were positive by PfHRP2-based RDTs. Although this phenomenon was not reported in the South and Central America studies, it may be due to close reaction of PfHRP2 monoclonal antibodies with PfHRP3. In vitro studies have confirmed the detection of parasites with deleted pfhrp2 on PfHRP2-based RDTs, but this has not been reported widely in field samples [pfhrp2/pfhrp3 deletion [In Kenya, one study has reported the presence of children . Curious samples . Becausedeletion .pfhrp2/pfhrp3 deletions have been widely reported in Central and South America, where most studies reported a higher prevalence of pfhrp3 (Lowest = 7.4% in French Guiana and Highest = 96.2% in Honduras) than of pfhrp2 (Lowest = 4.0% in Bolivia and Highest = 40.6% in Peru). Similar deletions were also observed for the upstream and downstream regions flanking pfhrp2 and pfhrp3 genes  [pfhrp2 exon1-2 was 0.10    to a max0, 0.04) . Further6, 0.17) A. The heectively B-G.pfhrp2/pfhrp3 deletions [P. falciparum by two expert microscopists and PCR. This is followed by a confirmatory evidence of deletion through PCR amplification of exon 2 of pfhrp2 and pfhrp3 and the region across exon 1 and 2 of both genes.Cheng et al.(2014) published a set of recommendations for reporting eletions . These rmsp1 and msp2 to rule out negative results due to low-quality DNA or PCR failure. We used these recommendations to evaluate which studies conformed to this protocol. All studies conducted in South America, Central America, and Asia conformed to these recommendations for reporting pfhrp2/pfhrp3 deletions including PCR analysis of the flanking genes, performing RDTs on the suspected samples, and functional analysis through PfHRP2 ELISA. These recommendations are also in agreement with the recently published WHO guidelines on reporting pfhrp2/pfhrp3 deletions [pfhrp2/pfhrp3 gene deletions.For quality control, it is also recommended to include PCR amplification of single copy genes such as eletions  and as spfhrp2 (Lowest = 1.5% in Mozambique and Highest = 62.0% in Eritrea) and pfhrp3  did not include RDT results [Unfortunately, of the eight studies conducted in Africa, only two followed these guidelines. Most reported on the prevalence of  Eritrea ,62,63,64 results .pfhrp2 and pfhrp3 are not well characterized, they have been observed [pfhrp2 and pfhrp3 gene deletions. As shown in pfhrp2/pfhrp3 deletions. This may affect the validity of the claims made [Although the upstream and downstream flanking genes for observed , and as ims made ,71.pfhrp2 and pfhrp3 genes has revealed extensive variability and different haplotype profiles in parasites from different geographical areas, and this may influence the performance of RDTs [PfHRP2 in plasma, which is the basis for parasite detection on RDTs [Apart from gene deletions, sequencing of the  of RDTs ,73. For  of RDTs ,74,75. T on RDTs .The extensive decline in malaria witnessed over the last two decades is attributed to improved diagnosis, effective treatment, and monitoring. The details of the decline are captured in the WHO Global Initiative for malaria control referred to as T3  which provided a framework for malaria control and elimination in malaria-endemic countries . This inpfhrp2 and/or pfhrp3 may negatively impact the success of RDTs in malaria diagnosis and could present a major threat to the progress made in the fight against malaria [pfhrp2 depleted parasites is not clearly defined, mathematical modeling has suggested that the use of PfHRP2-based RDTs over the last decade favored the survival of parasites lacking pfhrp2 genes. The model also suggests that selection pressure is high in areas where the prevalence of malaria is low, and the treatment of cases is based on RDT diagnosis [PfHRP2 and PfHRP3 antigens and the observed cross-reaction of monoclonal antibodies used in the RDTs, it would be expected that PfHRP3 would compensate for the deletion of pfhrp2. However, because PfHRP3 is not abundantly produced, this is likely to be beneficial only at high parasitemia [Recent evidence of false-negative RDTs due to the emergence of parasites lacking  malaria ,49, and iagnosis . This isiagnosis ,80. Becaasitemia . pfhrp2 depleted parasites are likely to go undetected [pfhrp2 deleted malaria parasites. The decline in malaria transmission that has been witnessed in most areas means that regions previously considered endemic may not experience as high a number of malaria cases as before, and most infections will likely be characterized by low-density parasitemia. If the model proposed by Watson is correct, this will increase the number of missed diagnosis by both microscopy and RDT; therefore, individuals harboring detected . Consequdetected . In peripfhrp2/pfhrp3 deleted parasites mostly in areas that have had a history of P. falciparum malaria. This could become a big challenge, especially with the increased use of PfHRP2-based RDTs. In sub-Saharan Africa, where malaria is still endemic, the extent of pfhrp2/pfhrp3 gene deletions is underreported [pfhrp2/pfhrp3 deleted parasites and the impact they have on malaria diagnosis.This review demonstrates the emerging threat of reported ,81. Ther


Malaria is one of the leading causes of mortality and morbidity in tropical and subtropical regions. The bulk of the global malaria burden is in sub-Saharan African countries, including Ethiopia. Malaria adversely affects the health of the peoples as well as the economic development of many developing countries including Ethiopia. I2), and time series analysis. The presence of publication bias was evaluated using funnel plots and Egger's regression test.  This review article was reported according to PRISMA guidelines. Related published articles were searched from online public databases, such as PubMed, Google Scholar, and ScienceDirect. The search approach used to retrieve related articles were “prevalence,” “malaria,” “adults,” and “Ethiopia.” The quality of articles was assessed using Joana Brigg's Institute (JBI) critical appraisal checklist. The meta-analysis was computed using STATA version 14. The pooled prevalence estimates with 95% confidence interval were analyzed using a random-effect model, and the possible source of heterogeneity across studies was indicated through subgroup analysis, inverse of variance  (16.17%) followed by Oromia Regional State (13.11%) and Amhara Regional State (12.41%).  Plasmodium parasites ) AND malaria) OR malaria [MeSH Terms]) (AND adults) OR adults [MeSH Terms]) AND Ethiopia) OR Ethiopia [MeSH Terms]). The repeated articles were excluded in the systematic and meta-analysis review. The software EndNote version X5  was used to arrange references and remove repeated references.Studies published in the peer-reviewed journals, which reports the prevalence of malaria among adults, were included. All studies were original research articles published in English and contained the basic information concerning sample size, diagnostic methods, prevalence, and status of malaria infection among adults in different parts of Ethiopia. Moreover, studies that have been carried out in health institution and community-based settings, symptomatic or asymptomatic individuals, and coinfections  as far as they reported malaria among adults were included this review.Studies conducted among all age groups, pregnant women, children, unknown malarial detection methods, unknown sample size, and lack of clear figure about infected cases were excluded from this review.Two authors (FA and TH) individually conducted a search in PubMed, Google Scholar, and ScienceDirect using the keywords. The searched articles were screened by the title and abstract to transfer the articles in the full-text review. The quality of articles was assessed using Joana Brigg's Institute (JBI) critical appraisal checklist for simple prevalence . The twoP. falciparum, the prevalence of P. vivax, prevalence of mixed infection, and type of the diagnostic method used.Data extraction protocol was developed by two authors (FA and TA) and evaluated by EN and MW. This extraction protocol consists of name of the first author, year of publication, study area (region), study group, study design, sample size, prevalence of malaria, prevalence of I2) [I2 values of 25%, 50%, and 75% were assumed to represent low, medium, and high heterogeneity, respectively. Due to the observed high heterogeneity across studies, we used a random effect model to assess pooled estimate. The analysis between the subgroups was carried out regarding the type of cases. Small study effect and publication bias across studies were evaluated by the funnel plot symmetry subjectively and Egger's regression test objectively.Eligible primary studies were extracted, entered into Microsoft Excel, and then exported to STATA version 14. Forest plots were used to estimate the pooled effect size and effect of each study with their confidence interval (CI) and to provide a visual image of the data. The degree of heterogeneity between the included studies was evaluated by the inverse of variance (I2) . I2 valuPlasmodium parasite. Finally, only eight full-text articles were screened for eligibility and included in the final quantitative meta-analysis   across studies was observed in the analysis.Eight articles were selected for this systematic review and meta-analysis, which were used to estimate the pooled prevalence of malaria among adults. The minimum prevalence of malaria was 4.3% observed in Oromia Regional State of different hospitals and health center , while tSubgroup analysis based on types of malaria cases showed that the prevalence of malaria among symptomatic and asymptomatic adults was 15.34% (95% CI: 4.66–26.01) and 11.99% (95% CI: 5.92–18.07), respectively . SimilarPlasmodium parasite species was compared between studies among Ethiopian adults. All of the studies [P. vivax and P. falciparum infection, while the prevalence of mixed infection was reported from seven studies. The overall pooled prevalence of Plasmodium species was as follows: P. falciparum (6.48%) (95% CI: 4.13–8.82), P. vivax (5.74%) (95% CI: 3.53–7.95), and mixed infection (0.47%) (95% CI: 0.09–0.85)  .pvalue = 0.262) indicated there was no evidence of publication bias across studies.The presence of publication bias was evaluated subjectively using funnel plots symmetry and objectively using Egger's regression test. The symmetrical distribution of the funnel plot indicated the absence of publication bias across studies . The resThe trend in malaria prevalence among included studies did not uniformly change within the ten years (2011–2018). From 2011 to 2014, the prevalence was characterized by a slight change in either direction (right and left). On the other hand, from 2016 to 2018, the prevalence showed an abrupt decrement and increment compared to the previous years .Plasmodium infections in some areas [Despite the declining of malaria in Ethiopia, the disease is still a major public health concern, and it is one of the leading causes of morbidity and mortality among adult populations , 35, espme areas . This mime areas .The current systematic review and meta-analysis was conducted using eight full-text articles to determine the pooled prevalence of malaria among adults in Ethiopia. Malaria causes serious complications in human, such as severe anaemia, acute renal failure, hypoglycemia , loss ofThis systematic review and meta-analysis study showed that the pooled prevalence of malaria among adults in Ethiopia was 13.61%. This is much greater than the 2015 malaria indicator survey among the general population of the country, which results in 1.2% and 0.5% malaria prevalence by RDT and microscopy tests, respectively . This stThe comparative decrement of malaria prevalence in the current study might be related to life style change, geographical area, economic status, the type of malaria diagnosis methods used, and the time at which the study conducted . In addiSubgroup analysis based on types of malaria cases showed that the prevalence of malaria among symptomatic and asymptomatic adults were 15.45% and 11.98%, respectively. The difference in the prevalence of malaria between the two groups is associated with parasite load; there might be higher parasite load among symptomatic cases than asymptomatic cases . SimilarThe trend of malaria prevalence among studies included in this review did not uniformly change within the ten years (2010–2019) . HoweverP. falciparum (6.48%) and P. vivax (5.74%) parasites was found in proportions of 48.6% and 47.5%, respectively. The result of this study showed that P. falciparum and P. vivax have nearly equal contributions to the disease, malaria. This is similar to a study conducted in Batu town, Oromia Regional State [P. falciparum proportion is greater than those of P. vivax in Ethiopia [P. vivax is higher than P. falciparum [P. falciparum and P. vivax infections in our study indicate a shift in the trend of species composition in Ethiopia.The overall pooled prevalence of al State . On the Ethiopia , 42, 45.lciparum –48. AlmoMalaria is an important public health problem among adult individuals in Ethiopia. This study summarized the prevalence of malaria among adult in Ethiopia that can be an input for policymakers. Despite this, the present study has few limitations. First, a small number of articles included in this systematic review and meta-analysis could affect the pooled prevalence estimate. Second, almost all of the included studies were cross-sectional studies, and due to such reasons, the outcome variable could be affected by other confounding variables. Third, half of the included articles were obtained from the Oromia Regional State, whereas the other half was found to be in Amhara Regional State and SNNPR. This unequal distribution of articles throughout the country may affect the outcomes of this study. Fourth, no study obtained from Afar, Benishangul Gumuz, Gambella, Harari, and Tigray regions. These aforementioned limitations might affect the results reported in this review regarding the overall prevalence of malaria among adults in Ethiopia.The current systematic review and meta-analysis showed that the pooled prevalence of malaria among adults was found to be greater than the general population and is nearly equal to pregnant women. Therefore, the existing prevention and control measures such as health education, usage of LLIN and application of IRS for controlling the vector, early diagnosis and treatment, and adaptation of artemisinin-based therapies should be adopted. In addition to this, Ethiopian public health institution and other responsible bodies should focus on the substantial reduction of mosquito-breading sites through community participation. Moreover, monitoring and mapping the distribution of insecticide resistance is appropriate in order to develop new vector control strategies.


Cholangiocarcinoma (CCA) is a rare biliary-duct malignancy with poor prognosis. Recently, the presence of the human polyomavirus 6 (HPyV6) has been reported in the bile of diverse hepatobiliary diseases, particularly in the bile of CCA patients. Here, we investigated the presence of novel HPyVs in CCA tissues using diverse molecular techniques to assess a possible role of HPyVs in CCA. Formalin-Fixed Paraffin-Embedded (FFPE) tissues of 42 CCA patients were included in this study. PCR-based screening for HPyVs was conducted using degenerated and HPyV-specific primers. Following that, we performed FISH, RNA in situ hybridization (RNA-ISH), and immunohistochemistry (IHC) to assess the presence of HPyVs in selected tissues. Of all 42 CCAs, 25 (59%) were positive for one HPyV, while 10 (24%) CCAs were positive for 2 HPyVs simultaneously, and 7 (17%) were negative for HPyVs. Of the total 35 positive CCAs, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 as confirmed by sequencing. The presence of viral nucleic acids was confirmed by specific FISH, while the RNA-ISH confirmed the presence of HPyV6 on the single-cell level. In addition, expression of HPyV7, HPyV6, and MCPyV proteins were confirmed by IHC. Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect bile duct epithelium, hepatocytes, and CCA cells, which possibly suggest an indirect role of these viruses in the etiopathogenesis of CCA. Furthermore, the observed hepatotropism of these novel HPyV, in particular HPyV7, might implicate a role of these viruses in other hepatobiliary diseases. Cholangiocarcinoma (CCA) is a rare but very aggressive biliary duct neoplasia with a very poor prognosis . The etiForty-two formalin fixed and paraffin embedded (FFPE) tissue specimens of CCA patients collected at the Department of Pathology, Maastricht University Medical Centre+ (Maastricht Pathology Tissue Collection number: 2015-13) were included in this study. The study was approved by the Medical Ethics Review Committee of the Maastricht UMC+, the Netherlands (2019-0977), all tissue resections were collected and studied in accordance with the protocol of the Dutch Code of Conduct for Observational Research with Personal Data (2004) and Tissue .Of these 42 CCA patients, 25 (60%) were male and 17 (40%) female. The mean age was 65.2 years (range: 29–85 yrs.). Three experienced pathologists  reviewed the histopathological and cytopathological features, and a consensus diagnosis of CCA subtypes was established. The recognition of the tubular non-neoplastic bile duct epithelium and neoplastic CCA cells has been performed by the above-mentioned pathologists.DNA was isolated from all 42 FFPE tissues using techniques as previously described . In brieIn order to screen for the presence of HPyVs DNA, we modified the degenerated primers as recently published by Chan et al. . The priThe first nineteen isolated DNAs were screened for the presence of HPyVs DNA using degenerate primers, followed by sequencing of the obtained PCR products. DNA sequences were compared with the reference sequences of the National Center for Biotechnology Information (NCBI) Entrez Nucleotide database using the NCBI Blast program.In addition to the degenerated PCR technique, the specific PCRs targeting different gene regions of HPyV7, HPyV6, and MCPyV were performed using the protocols as previously described . We usedAll FISH procedures were carried out as described previously ,17,18,19The FISH slides were analyzed by at least three members  and the results of all cases were discussed together with all authors according to the criteria as previously described by Hafkamp et al. . In addi® HD Red 2·5 Kit  according to the manufacturer’s instructions.The RNAscope assay using complementary RNA probes for the localization of specific expression of HPyVs mRNA was applied to selected cases: A specific probe for both HPyV6 and 7 LTAg genes was designed by Advanced Cell Diagnostics (ACD). A total of 20 pairs of 50 bp pooled probe designed for the HPyV7 target region between 3688 and 4876 bp . Another 20 pairs of RNA probes for the HPyV6 target region 3786–4898 bp . The sections were pretreated using the RNAscopeA probe for human peptidylprolyl isomerase B (Hs-PPIB) mRNA expression was used as a positive control, while a probe for bacterial dihydrodipicolinate reductase gene (DapB) expression was used as a negative control. First, Hs-PPIB and DapB probes were applied to FFPE HeLa cells to validate the pretreatment and hybridization conditions according to ACD protocols . In addiThe following antibodies and dilutions were used: the monoclonal antibodies 2T10 directed against HPyV7-sTAg and 1T1 directed against HPyV6-sTAg were provided by C.Buck, NCI, Bethesda, MD, USA, and used at a 1:100 dilution in combination with the EnVision-FLEX™ visualization Kit  according to standard protocols . 2T10-AbIn addition, the CM2B4 monoclonal antibody was used to detect LT-antigen expression of MCPyV, which is more sensitive and specific than 2T10 and 1T1 Ab as described by Moshiri et al.  . The proHPyV7-positive thymoma tissues were used as previously published . KeratoaThe immunohistochemistry stainings were scored either microscopically or using digital slides. The whole tissue sections were evaluated for the presence of positive nuclei and cytoplasm in hepatocytes, large bile duct, portal tracts, and neoplastic CCA cells. The percentage of positive cells was assessed. Inflammatory infiltrate was also evaluated if it was present. The intensity of HPyVs staining was evaluated and scored as strong (+++) if the positive nuclei were seen at 100× magnification, moderate (++) if the positive nuclei were seen at 200× magnification and weak (+) if positive nuclei were seen at 400× magnification.In all subanatomic histological compartments , the quantitative analysis of HPyV7 IHC positive cells was evaluated using morphometry by  on digital slides. The slides were scanned with the VENTANA iScan-HT slide scanner  at 400× magnification. The morphometry was performed by calculating positive nuclei in the epithelial lining of the portal bile ducts, hepatocytes, and tumor cells in the hotspots in 5 photos at 400× magnification . The average of positive nuclei in the five images was calculated, and the results were present as a number of positive cells per HPF.p-value of <0.05 was considered to be statistically significant. The cohort was used to build the survival model. For feature selection, we first removed correlated features (Pearson’s r > 0.90), using the publicly available Caret package [The overall survival (OS) curve was estimated using the Kaplan–Meier method, and differences between HPyV-positive and -negative CCA cases were evaluated using a log rank test. OS was defined as the time between the date of tumor biopsy and date of death. Follow-up data for 39 (93%) patients were available, of which two patients were excluded due to the lack of survival data, only iCCA and pCCA were selected. Statistical analysis was performed with R-studio, and a  package . The rem package . The hazAll isolated DNAs of the FFPE blocks revealed sufficient DNA quality with PCR products > 300 bp based on the results of the specimen control size (SCS) ladder PCR . A conseWe used the MCPyV-positive MKL-1 cell line and diverse polyomaviruses (PyVs) plasmids as positive controls for PCR with the modified degenerated primers. Accordingly, the modified degenerated primers were able to amplify PyVs sequences from MCPyV, HPyV6, 7, 9, NJPyV, WUV, CaPyV, and BPyV plasmids as well as the MKL-1 cell line , as confUsing HPyV7-specific PCR for all CCA cases of the cohort, 62% (26/42) of CCA tissues were tested positive for the LTAg in comparison to 26% (11/42) CCAs which were tested positive for the sTAg DNA-PCR b Supple. In contIn summary, the results of all PCR approaches revealed that 59% (25/42) of CCA tissues were positive for one of the HPyVs, while 10 (24%) CCA tissues were positive for 2 HPyVs, and 7 (17%) CCA tissues were negative for any HPyVs. Of the total 35 positive CCA tissues, 19 (45%) were positive for HPyV7, 4 (9%) for HPyV6, 2 (5%) for Merkel cell polyomavirus (MCPyV), 8 (19%) for both HPyV7/MCPyV, and 2 (5%) for both HPyV6/HPyV7 c,d. CombSelected FFPE tissues chosen for assessing the FISH pretreatment with the alpha satellite (centromeric) DNA probe quality revealed intense FISH signals in all parenchymal and non-parenchymal liver cells. Thus, the protocol was optimal and was used for subsequent HPyV FISH analyses without modifications .Twelve CCA tissues were subjected to HPyV7-specific FISH analysis and four of them revealed specific nuclear signals in CCA tumor cells as well as non-neoplastic bile duct epithelium and hepatocytes . HPyV7 DAll three HPyV6 DNA PCR positive cases showed specific nuclear HPyV6 DNA signals after FISH, although the dots were observed more often in the hepatocytes with normal morphology than in the CCA cells b. AdditiThe RNAscope technique was applied on 5 CCA tissues  and six showed moderate to weak nuclear expression in five–ten percent of the tumor cells a. The exMoreover, the number of HPyV7-positive nuclei in hepatocytes, bile duct epithelium, and tumor cells was counted in the hotspots in 5 high power fields  using morphometric analysis . Thus, tTwelve of the selected fourteen CCA tissues were tested positive for HPyV6-sTAg IHC expression. However, more CCA tissues revealed HPyV6-IHC positivity than PCR due to the fact that 1t1 antibodies are well known to have cross-reactivity with HPyV7 and MCPyV. The intensity of the HPyV6-IHC staining was weak to moderate with a maximal 10% of positive cells b. No sTASix CCA tissues were tested for MCPyV-LTag expression by IHC, five of which showed nuclear staining  c. Three p = 0.726), HPyV6 (p = 0.366), and MCPyV (p = 0.953) in iCCA and pCCA. The hazards ratio and the p-value are reported in Cox regression data  and HPyV7 , HPyV7 DNA was detected much more frequently in 29 (69%) of the CCA tissues. Of interest, some of the cases that tested HPyV7 DNA positive by degenerate LTAg PCR were negative by specific HPyV7 DNA-PCR targeting sTAg. However, assessing the whole cohort with a specific LTAg HPyV7 DNA-PCR within the conserved region which is targeted by the degenerated primers revealed a good agreement . This ap%, HPyV7 An explanation for the discrepancy between high HPyV7 prevalence found by us and high HPyV6 prevalence found by Chan et al. might be the primary source of the DNA, i.e., primary CCA tissues versus bile fluids of CCA patients, respectively, or due to variation in the ethnical and geographical distribution of both cohorts. Recent studies have assessed HPyVs serum antibody levels across a range of age groups including one from the Netherlands. HPyV7 and HPyV6 were found to be highly seroprevalent in all age groups ,31,32,33In addition to HPyV7 specific DNA PCR, we were able to visualize the presence of HPyV7 by FISH and IHC. The FISH results were in good agreement with the PCR results and allowed the identification of the virus at the single cell level within the histomorphological context. FISH revealed the presence of HPyV7 within non-neoplastic hepatocytes and bile duct epithelium as well as CCA tumor tissue a. HoweveUnfortunately, we were unable to detect specific mRNA signals for HPyV7 RISH due to technical reasons, which could be explained by two possibilities: the background ratio in the selected tissues made an interpretation of specific HPyV7 mRNA signals impossible; or the selected target sequences of the HPyV7 probe need to be adapted.The above findings indicate that HPyV7 is most probably not directly related to the etiopathogenesis of CCA. However, the presence of HPyV7 in only a part of the tumor cells may also have been caused by technical restrictions of the FISH and IHC assays. The detection sensitivity of these assays is dependent on optimal pre-analytic tissue processing and preservation of nucleic acid quality and protein epitopes, which sometimes can be limited in FFPE tissues.In the underlying study, a relatively low 14% prevalence of HPyV6 was detected in CCA tissues by HPyV6 specific PCR, which is lower than the 27% reported by Chan et al. in the bile fluid of CCA patients. By using the RNAscope technique, we were able to demonstrate HPyV6 transcripts on the single cell level in both non-neoplastic hepatocytes and CCA cells. This is in line with the DNA FISH and sTAg IHC results. The IHC results, however, need to be interpreted with care, since we also detected the HPyV6 sTAg expression in HPyV6 PCR-negative but HPyV7- and MCPyV- PCR positive cases, which is most likely due to the known cross-reactivity of this antibody with HPyV7 and MCPyV .MCPyV was found in 24% of CCAs by both degenerated and specific DNA PCR, which is consistent with previous reports ,37. LoyoThe contribution of HPyV 7 to CCA carcinogenesis cannot be definitely elucidated by the present data. However, it remains speculation in as much as HPyV7 possibly contributes to CCA carcinogenesis. This might be based on a direct HPyV7-interaction and subsequent deactivation of p53, as recently reported by Rozenblatt-Rosen et al. , or alten = 42). Of course, it would be of clinical interest to further elucidate possible other correlations between these HPyVs and clinical endpoints of CCA.There are some limitations to our study. These limitations are mainly due to the rarity of CCA which also explains the sample size of our patient cohort (Our results strongly indicate that HPyV7, HPyV6, and MCPyV infect non-neoplastic hepatocytes, bile duct epithelium, and CCA lesions. Although all three HPyVs are hepatotropic, HPyV7 was more frequently detected in both neoplastic and non-neoplastic cells in our CCA subset compared to HPyV6 and MCPyV. To the best of our knowledge, this is the first study to report the presence of HPyV7, HpyV6, and MCPyV on the single cell level in liver tissues of non-neoplastic bile duct epithelium and hepatocytes and CCA. We used diverse molecular techniques to assess the presence of these viruses. Although our results might point to indirect role of these HPyVs to CCA carcinogenesis, possibly by inflammation, the role of these HPyVs in the etiopathogenesis of CCA needs to be further elucidated. Furthermore, the frequent finding of HPyVs, especially HPyV7 in liver tissues might also point to a possible role of HPyV7 in other hepatocellular diseases.


Plasmodium vivax, are found in native communities of The Rio Santiago District, Condorcanqui Province. In 2019, more than one thousand malaria cases were reported, with an unusual number of Plasmodium falciparum autochthonous cases. The present study aims to report this P. falciparum outbreak while describing the epidemiology of malaria and the risk factors associated in the native communities of Amazonas, Peru.Malaria remains a serious health threat in the Amazonas Region of Peru and approximately 95% of the cases, mainly The DIRESA-Amazonas in collaboration with the Condorcanqui Health Network and the Institute of Tropical Diseases of the UNTRM carried out a malaria Active Case Detection (ACD III) between January 31st and February 10th of 2020. A total of 2718 (47.4%) individuals from 21 native communities grouped in eight sanitary districts, were screened for malaria infections. Each participant was screened for malaria using microscopy. Follow-up surveys were conducted for all malaria positive individuals to collect socio-demographic data. Spatial clustering of infection risk was calculated using a generalized linear model (GLM). Analysis of risk considered factors such as gender, age, type of infection, symptomatology, and parasitaemia.P. falciparum index case was imported from Loreto and later spread to other communities of Rio Santiago during 2019. The ACD III reported 220 (8.1%) malaria cases, 46 P. falciparum, 168 P. vivax and 6 mixed infections. SaTScan analysis detected a cluster of high infection risk in Middle Rio Santiago and a particular high P. falciparum infection risk cluster in Upper Rio Santiago. Interestingly, the evaluation of different risk factors showed significant associations between low parasitaemia and P. falciparum asymptomatic cases.The study suggests that the P. falciparum outbreak in native communities of Condorcanqui, Amazonas. Timely identification and treatment of symptomatic and asymptomatic cases are critical to achieve malaria control and possible elimination in this area. However, the current malaria situation in Condorcanqui is uncertain, given that malaria ACD activities have been postponed due to COVID-19.This is the first report of a  Plasmodium falciparum, representing a risk for severe malaria and mortality. It is worth noting that since 2015 only four P. falciparum imported cases were reported. In 2018, 705 malaria cases were reported and this number increased to 1843 in 2019, including 807 new P. falciparum cases  of the Universidad Nacional Toribio Rodríguez de Mendoza (UNTRM) collaborated with these institutions during ACDIII activities between January 31st and February 10th of 2020 and the data collected was analysed in this manuscript. Activities were performed at the community centres and a door-to-door strategy was adopted for those families who did not attend the open call. This study was performed as part of the surveillance activities approved by the DIRESA-Amazonas and the National Ministry of Health.The Regional Directorate of Health-Amazonas (DIRESA-Amazonas) in collaboration with the Condorcanqui Health Network performed malaria active case detection (ACD) activities, in the Rio Santiago District, Condorcanqui Province, Amazonas Region of Peru, in response to the Informed consent was obtained from “Apus” (chiefs of the communities) as well as from all individual participants included in the study. For underage children, informed consent of parents or legal guardian was obtained.2 [Condorcanqui Province is located in the northern jungle of the Amazonas Region of Peru Fig. , and it 2 , with an2 .Fig. 2MaThe rugged relief of this region has an extensive fluvial network constituted by the Marañon River and its tributaries Cenepa, Nieva, and Santiago, in addition to a large number of streams of different flows and sizes. Condorcanqui has a humid tropic climate with temperatures that can reach 35 °C, annual average rainfall around 4800 mm, and relative humidity above 90%. The rainy season occurs between October to December, but it could last until May [A total of 2718 individuals  were screened for malaria infections in 21 native communities. These communities were grouped in eight “sanitary districts”  according to their corresponding health post  were available only for people reporting symptoms. Microscopy results were given the next day and all positive individuals were treated according to the Peruvian Ministry of Health guidelines for malaria cases .A registry of socio-demographic variables such as the name of the community, age, gender, and presence of symptoms was recorded for coded positive malaria cases with no personal identifiers. Data of previous malaria cases in Condorcanqui was obtained from the DIRESA-Amazonas database with no names or personal identifiers.Thick and thin blood smears were stained with Giemsa. The slides were examined under compound microscopes (Olympus) at 1000× magnification with oil to detect malaria parasites. Ring, trophozoite, or gametocyte stages were identified and assessed for parasite density using the semi-quantitative method according to the Peruvian Ministry of Health guidelines . The micSD Bioline Malaria Ag P.f/P.f/P.v RDTs were available only for patients who presented symptoms. Fifteen percent of the positive samples (n = 35) were randomly collected in Whatman™ Protein Saver cards 903 and confirmed by qPCR at IET for quality control according to a previously reported protocol [Plasmodium vivax cases were treated with chloroquine and primaquine for 7 days and positive P. falciparum cases with mefloquine and artesunate for 3 days with additional primaquine (0.75 mg/kg) the last day of treatment. Mixed infections were treated with mefloquine, artesunate, and subsequently primaquine (0.5 mg/kg/day) for 7 days. Children under 12 years of age were treated according to their weight [Positive r weight .Plasmodium species observed by microscopy; mixed infections were reported if both P. vivax and P. falciparum were present. Parasitaemia was classified as “low” if less than 40 parasites were found in at least 100 fields and “high” if more than 40 parasites were found. Symptomatic individuals were defined as those who presented any of the following symptoms at the time of taking the blood sample: fever, headache, body pain, shivering, nausea, vomiting, and/or abdominal pain. Asymptomatic individuals were defined as persons with confirmed infection showing no symptoms of infection or disease.Malaria cases were classified into six age groups defined as: children under 5 years old, children (5–11 years old), teenagers (12–17 years old), youngsters (18–29 years old), adults (30–59 years old) and seniors (over 60 years old). Type of infection was recorded according to the P. falciparum), and parasitaemia (low) with the dependent variables, symptomatology (asymptomatic), type of infection  and parasitaemia (low).Prevalence of malaria positive cases with a 95% confidence interval (CI) was calculated for each sanitary district and according to species. Omnibus test (Chi-square) was used to determine the association between location (sanitary districts) and presence of malaria . Then, aAdditional bivariate cross-tabulations Pearson Chi-Square analysis with bilateral asymptotical significance  and Wald test from logistic regression were performed to examine these associations with 95% confidence. All the statistical analyses were based on Agresti, 2007 , and werP. falciparum and P. vivax). To analyse the effect of the surveillance in these communities, heat maps of malaria incidence per 1000 habitants, considering only passive surveillance before and after ACD were also included.A spatial analysis to detect clusters with high and low risk of malaria infection was conducted using the SaTScan software (version 9.6 64-bit). A Bernoulli model was applied to evaluate the spatial distribution in sanitary districts with positive malaria cases relative to the control group (negative cases). Clusters were detected with a maximum spatial cluster size of 50% of the population at risk  and a ciP. falciparum were reported in Condorcanqui. The last case reported was a man visiting from the Shinguito community of Loreto (57 km away from Condorcanqui). Five months later, in January of 2019, the first two P. falciparum autochthonous cases were found in Soledad, a 3-year-old girl, and an 11-year-old boy. Then, sporadic cases were reported in the sanitary districts of Ayambis, Soledad, Chapiza, Nauta, Cucuasa, and Chosica. However, an unusual increase in the number of P. falciparum cases was notified after epidemiological week 25 of 2019  and Soledad (5.2%) where P. falciparum infections were greater. The highest P. vivax prevalence was found in Yutupis (13.4%), followed by Chosica (12.1%), Chapiza (7.2%), and Nauta (6.8%). Also, 3.6% of the total malaria positive cases  were P. falciparum gametocytes carriers, and only one of them showed symptoms.A total of 220 malaria positive cases were found in the population screened by microscopy n = 2718, 47.4%). The prevalence of malaria in this area was 8.1%, from which Chosica (13.2%) and Yutupis (13.4%) depicted the highest prevalence  than males (41.8%) and according to the age distribution, the number of malaria cases was higher in children between 5 and 11 years old (30.5%)  were P. vivax. Only one asymptomatic mixed infection was reported. However, it is worth noting that from all P. falciparum cases, 80.4% reported no symptoms compared to 55.4% of P. vivax cases.From all positive cases, 76.4% were diagnosed with ears old 0.5% . The estimated parameter of the generalized linear model with binomial distribution indicated significant differences among all sanitary districts (p < 0.001) with a higher probability of infection in Ayambis, Nauta, Chapiza, Chosica, and Yutupis compared to Cucuasa and Guayabal . Furthermore, significant associations between adults and P. falciparum infections , and between low parasitaemia and P. falciparum infections  were also found  and between P. vivax and P. falciparum represents a major public health problem in Peru. Most of the control efforts are focused in Loreto, where 95.6% of all cases are reported [P. vivax cases during the past 2 years with the recent emergence of P. falciparum cases in 2019 [P. falciparum imported case from Shinguito, Loreto, was reported. This community is 57 km apart from the community of Soledad in Condorcanqui, where the first two P. falciparum autochthonous cases were reported. These cases corresponded to symptomatic children that were taken to the health post; however, it is possible that infected adults did not report symptoms and remained unattended, contributing to the transmission of the disease. The following weeks, P. falciparum cases spread to Soledad neighbouring communities and other sanitary districts. Towards the epidemiological week 27 of 2019, a high number of cases was observed. This fact can be explained by the increase of travels to and from Loreto due to The San Juan Festival, which is a major event in the Amazon of Peru. This festival is held for one week with the main celebration date on June 24th, where people party and exchange goods. This could have increased the number of infections and the spread of P. falciparum to other communities such as Chapiza, Nauta, and Chosica. Moreover, Alianza Progreso, located within the sanitary district of Chapiza, is a centre of several social, commercial, and economical activities, where people from other communities stay after 6 pm , representing a hotspot for malaria transmission. Besides, climate changes caused by El Niño-Southern Oscillation could be related to the reemergence of malaria in this area over the years. Since the number of malaria cases increased in Condorcanqui, DIRESA-Amazonas organized several ACDs to control malaria and the spread of P. falciparum to other native communities.Malaria disease caused by reported . However in 2019 . In the P. falciparum and P. vivax presented different spatial distribution. The recent introduction of P. falciparum in Soledad can explain the presence of a high-risk cluster, while P. vivax is endemic in all Rio Santiago. Moreover, six mixed infections were reported, which represented a challenge for a correct diagnosis and treatment of patients.According to previous data from ACD I and II, the highest prevalence of malaria was reported in Chosica (19.1%), followed by Nauta 12.4%) and Chapiza (10.2%) [% and ChaP. vivax parasitaemia are linked to specific factors such as travel and occupation, which created a complex transmission dynamics in these communities [A previous study in riverine communities of Loreto determined that malaria prevalence and the micro-geographical heterogeneity of munities . These aIn Condorcanqui, descriptive analysis of positive cases exhibited a higher number in females than in males and the number of cases in children (5–11 years old) was also higher than in other age groups. The lack of complete sociodemographic data of negative cases hinders further statistical analysis of infection risk. Although a door-to-door strategy was considered for individuals who did not attend the open call, some men were working in the field and some declined their participation in the study which might generated a bias towards women participants.Nevertheless, it has been reported that in malaria-endemic zones, children and pregnant women are the groups at highest risk and the most affected due to poor socio-economic conditions and nutritional problems , somethiP. vivax cases were predominant, most P. falciparum cases in Condorcanqui were asymptomatic. Asymptomatic malaria infections hamper disease control. Under normal circumstances, these patients do not seek treatment and they become parasite reservoirs contributing to the malaria cycle in the population [On the other hand, although pulation .P. falciparum, asymptomatic cases, and adults (30–59 years old), consistent with previous studies [P. falciparum infection in adults may be due to their activities, such as farming, hunting, or fishing, being in close contact with mosquito breeding sites, and more exposed to infections than other age groups. Low parasitaemia cases represent a challenge to the correct diagnosis and according to previous studies, there are significant numbers of submicroscopic asymptomatic cases only detectable by qPCR given that these subjects retained control over the parasitaemia [Additionally, the evaluation of different risk factors showed significant associations between asymptomatic cases and low parasitaemia, these relative risks are in agreement with previous reports of endemic areas , 18, sug studies , 19–21. sitaemia . It is wsitaemia . Moreovesitaemia .Pfhrp2 and Pfhrp3 deletions have been previously reported in Peru [P. falciparum detection. Thus, it is important to improve malaria diagnosis in native communities of Condorcanqui using complementary strategies, such as microscopy and RDTs or molecular biology techniques, for an effective malaria control [Rapid diagnostic tests (RDTs) could be considered a complementary tool for prompt malaria diagnosis , especia in Peru  and thes control .P. falciparum treatment in 3 days is achievable compared to P. vivax treatment, which requires supervision for 7 days [P. vivax hypnozoites could be responsible for recurrences within 24 weeks [P. vivax control. Comparison of P. falciparum and P. vivax cases detected during passive surveillance before and after the four ACDs showed a significant decrease in P. falciparum prevalence, indicating a successful control of the outbreak; while P. vivax hotspots continued to be present . Continue surveillance of malaria in endemic regions is important to prevent severe malaria and deaths as a result of a co-infection with SARS-CoV-2.Although the number of malaria cases decreased after epidemiological week 20 of 2020, it has to be considered that the COVID-19 pandemic has heavily impacted the health services and the report systems of other diseases. The first COVID-19 case was reported in Condorcanqui towards the epidemiological week 19 of 2020, and by week 23, cases spread to other sanitary districts, reporting over 500 cases. It is possible that malaria infections are currently underestimated and that the increase of COVID-19 cases hinders not only P. falciparum outbreak, and the presence of asymptomatic cases. Results presented here are useful to understand malaria transmission in native communities and allow targeted activities for malaria surveillance and control in this area. In conclusion, it is important to improve diagnosis to detect as many asymptomatic and symptomatic cases as possible to promptly treat and prevent the spread of this disease to other communities of Amazonas, Peru. Moreover, the current malaria situation in Condorcanqui is worrying, since lack of follow-up can lead to the establishment of P. falciparum and syndemics with COVID-19.This article represents the first study describing the epidemiology of malaria cases in native communities of Condorcanqui, Amazonas, the first reported Additional file 1: Fig. S1. Malaria cases per sanitary district during 2019 and 2020. Major events such as the San Juan festival and ACDs  are indicated. First COVID-19 reports in Condorcanqui are also indicated. a.Plasmodium falciparum cases. b.Plasmodium vivax cases.Additional file 2: Table S1. Pearson chi-square (χ2) and contingency coefficient for positive malaria cases in Río Santiago.Additional file 3: Fig. S2. Heat map of Plasmodium falciparum incidence per 1000 habitants before and after all ACD. Maps show 21 weeks before ACD I (left) and 21 weeks after ACD IV (right). Maximum value: 254.Additional file 4: Fig. S3. Heat map of Plasmodium vivax incidence per 1,000 habitants before and after all ACD. Maps show 21 weeks before ACD I (left) and 21 weeks after ACD IV (right). Maximum value: 254.


The development of innovative diagnostic tests is fundamental in the route towards malaria eradication. Here, we discuss the sorting capabilities of an innovative test for malaria which allows the quantitative and rapid detection of all malaria species. The physical concept of the test exploits the paramagnetic property of infected erythrocytes and hemozoin crystals, the magnetic fingerprints of malaria common to all species, which allows them to undergo a selective magnetophoretic separation driven by a magnetic field gradient in competition with gravity. Upon separation, corpuscles concentrate at the surface of a silicon microchip where interdigitated electrodes are placed in close proximity to magnetic concentrators. The impedance variation proportional to the amount of attracted particles is then measured. The capability of our test to perform the selective detection of infected erythrocytes and hemozoin crystals has been tested by means of capture experiments on treated bovine red blood cells, mimicking the behavior of malaria-infected ones, and suspensions of synthetic hemozoin crystals. Different configuration angles of the chip with respect to gravity force and different thicknesses of the microfluidic chamber containing the blood sample have been investigated experimentally and by multiphysics simulations. In the paper, we describe the optimum conditions leading to maximum sensitivity and specificity of the test. Malaria, caused by Plasmodium parasites transmitted to people through the bites of Anopheles mosquitoes, is one of the most diffused infectious disease worldwide. The “Word Malaria Report 2019” reports 228 million new cases in 2018, mainly in the African Region (93%), Southeast Asia Region (3.4%), and Eastern Mediterranean Region (2%) [Conventional methods for malaria diagnosis are not suitable for on-field wide screening of the population. Optical microscopy examination, the time-honored gold standard for malaria, asks for an expert operator able to distinguish infected red blood cells (i-RBCs) in a blood smear using a good microscope. It is thus an operator-dependent method, requiring approximately 60 min and a laboratory setting . Rapid DTo fill the gap between the current technology and the need for a cheap, fast, and highly sensitive diagnostic tool suitable to wide screening in low-resource setting, the World Health Organization itself strongly recommends the development of novel RTDs with the same sensitivity of microscopy but with a reduced number of false positives and false negatives with respect to currently available lateral-flow devices.In this paper, we discuss the capability of a novel rapid diagnostic test under development to selectively detect infected red blood cells (i-RBCs) and free malaria pigment in a blood sample . It is wproduced . α-hemat the RBC , and it tal (HC) ,11,12. (1:1:8) ,14. Note (1:1:8) .Plasmodium falciparum, the adherence of infected erythrocytes containing late developmental stages of the parasite (trophozoites and schizonts) to the endothelium of capillaries and venules strongly reduces the concentration of i-RBCs detectable after staining [Interestingly enough, hemozoin has been used as malaria biomarker since the very beginning of microscopy analysis. The detection of free HCs in blood smears by expert microscopists can help to identify the stage of the infection, as large concentrations are expected after the cell membrane rupture, responsible for the febrile attack. Furthermore, free malaria pigment is the unique detectable marker for microscopy in case of sequestration. In particular for staining .Furthermore, hemozoin crystals exhibit interesting physical properties such as paramagnetism and optical dichroism. Paramagnetism is given by the central iron ion of hematin in ferric state Febehavior ,17,18. Abehavior ,23,24,25Our test is based on paramagnetic properties of malaria i-RBCs and HCs which allow their selective magnetophoretic separation, driven by an high magnetic field gradient, in a lab-on-chip platform. The infected blood corpuscles are concentrated at the surface of gold interdigitated electrodes thanks to to magnetic concentrators placed in closed proximity whereas healthy ones sediment under the action of gravity. Then, a change in resistivity, proportional to the amount of attracted particles (i-RBCs and HCs) is detected as an impedance variation.Here, we show that in the horizontal configuration  our system selectively detects only hemozoin crystals, while in the vertical configuration (chip surface perpendicular to the floor), also i-RBCs are sorted and detected. In particular, the impact on the detection efficiency of the angle Following a lab-on-chip approach, the test is based on a silicon microchip e that haThe microchip consists on an arrangement of Ni concentrators with a diameter of 40 ntrators f. With tntrators g are plaThe blood sample to be analyzed is dispensed on a glass slide where a polymeric confinement gasket, made of polydimethylsiloxane (PDMS) b with thr motion d allows Particle transport in a magnetophoretic system is influenced by many forces of different nature: (i) magnetic force due to magnetic field gradients, (ii) viscous drag, (iii) gravity, (iv) buoyancy, (v) random forces due to thermal kinetics, (vi) particle–fluid interactions, and (vii) interparticle effects such as Van der Waals force.The gravity force  a horizontal configuration with the chip surface parallel to the floor and facing downwards a, α=0∘; he floor b, α=90∘.tal case a, the matal case b, the maThe force exerted on a magnetic particle, assuming that the length scale over which the magnetic field varies is much bigger with respect to the size of the magnetic particle itself and that the magnetic susceptibility of the particle is constant, can be expressed as tic test .Assuming that the volume of a red blood cells is 25g·cm−3 ,30, the https://www.supermagnete.it/)) permanent magnets, in the shape of parallelepipeds with 6 × 25 × 25 mmThis field gradient is provided macroscopically by a system of external magnets a,b and mAs mentioned above, red blood cells can be chemically treated to change their magnetic susceptibility becoming very similar to the one measured on malaria-infected red blood cells. In particular, the treatment aims to transform the hemoglobin contained in the RBC from oxyhemoglobin (oxy-Hb), which is diamagnetic, to methemoglobin (met-Hb), which is paramagnetic. In order to cause this transformation, RBCs must be exposed to oxidizing drugs, like As anticipated above, depending on the configuration angle it is possible to tune the capture efficiency or achieve selectivity with respect to different corpuscles. Indeed, gravity force plays a fundamental role especially when guration a. In thiThe experiments involving hemozoin have been carried out using synthetic crystals  to obtain a total haematocrit of 4%. In case of data reported in guration e it is aTo summarize, the experimental results reported in The influence of the angle vity see a on the As explained in The ratio between the specific and non-specific signal, within the error bars, is maximum for 90 degrees. Thus this is the preferred configuration in order to minimize the impact of false positive results while maximizing the test sensitivity.The height of the microfluidic chamber δ in a, defineThese results are also confirmed by multiphysics simulations carried out with COMSOL Multiphysics 5.3a where each t-RBCs was modeled as a spherical particle with volume We can conclude that, according to the protocol adopted in this example, the best conditions for increasing the signal correspond to In this paper, we discussed some specific features of a novel system for malaria diagnosis, based on the selective magnetophoretic capture and electrical impedance detection of malaria infected red blood cells and free hemozoin crystals. We showed that the test can selectively detect free circulating hemozoin crystals or the combination of both hemozoin crystals and infected RBCs, exploiting the same chip but horizontal and vertical configurations, respectively. In the vertical configuration, we checked the influence of a fine tuning of the angle between the chip surface and gravity force around 90 degrees. We found that 90 degrees is the optimum angle allowing to achieve the largest ratio between the specific and non-specific signal, in order to minimize the impact of false positive results. The cell height also strongly influence the detection efficiency. It turns out that increasing the cell height does not allow to improve the signal at fixed concentration, mainly due to corpuscle sedimentation from the sample load to the start of the measurement, according to the measurement protocol currently used. These findings are highly relevant for future developments of the test, allowing to improve its limit of detection and reduce the impact of false positive results. Moreover, the capability of performing an automatic selective detection of hemozoin crystals and infected RBCs represent a unique feature in the arena of hemozoin based tests for malaria, which could be of great help for on-field reliable assessment of the sickness evolution.Schistosoma mansoni worms [The proposed diagnostic test, in the present form, cannot easily distinguish between real malaria-infected red blood cells and red blood cells of a patient affected by methemoglobinemia, containing a lot of methemoglobin as in treated RBCs used for the present study. Even though the incidence of methemoglobinemia with respect to malaria could be negligible in endemic zone, the interference on the test results arising from other hematic pathologies is a crucial point which will require further investigation to assess the test specificity. On the other hand, our approach could be used to diagnose also other diseases in which organisms, such as ni worms ,34, feedhttp://hdl.handle.net/11311/1126859).“Apparatus for the quantification of biological components dispersed in a fluid” (


Despite potent anti-malarial treatment, mortality rates associated with severe falciparum malaria remain high. To attempt to improve outcome, several trials have assessed a variety of potential adjunctive therapeutics, however none to date has been shown to be beneficial. This may be due, at least partly, to the therapeutics chosen and clinical trial design used. Here, we highlight three themes that could facilitate the choice and evaluation of putative adjuvant interventions for severe malaria, paving the way for their assessment in randomized controlled trials. Most clinical trials of adjunctive therapeutics to date have been underpowered due to the large number of participants required to reach mortality endpoints, rendering these study designs challenging and expensive to conduct. These limitations may be mitigated by the use of risk-stratification of participants and application of surrogate endpoints. Appropriate surrogate endpoints include direct measures of pathways causally involved in the pathobiology of severe and fatal malaria, including markers of host immune and endothelial activation and microcirculatory dysfunction. We propose using circulating markers of these pathways to identify high-risk participants that would be most likely to benefit from adjunctive therapy, and further by adopting these biomarkers as surrogate endpoints; moreover, choosing interventions that target deleterious host immune responses that directly contribute to microcirculatory dysfunction, multi-organ dysfunction and death; and, finally, prioritizing where possible, drugs that act on these pathways that are already approved by the FDA, or other regulators, for other indications, and are known to be safe in target populations, including children. An emerging understanding of the critical role of the host response in severe malaria pathogenesis may facilitate both clinical trial design and the search of effective adjunctive therapeutics. Mortality and morbidity rates associated with falciparum malaria infection remain high. The World Health Organization (WHO) estimated that malaria accounted for 405,000 deaths in 2018 –5. The GSM is a multi-organ syndrome resulting from a complex interaction between both pathogen and host determinants, and its pathophysiology is yet to be fully understood . HoweverIn SSA, there are challenges in the early recognition and triage of SM, with as few as 10% of malaria cases appropriately triaged for care and < 30% of SM cases diagnosed and treated promptly, resulting in increased mortality and brain injury in survivors , 13. WHOPlasmodium vivax and Plasmodium knowlesi infections [It is important to re-evaluate WHO criteria to include emerging insights of SM pathogenesis and new aspects of SM epidemiology. Additionally, complementing WHO criteria with prognostic biomarkers could help identify high-risk patients that would most benefit from RCTs. Histidine-rich protein-2 (HRP-2), lactate, C-reactive protein (CRP) and procalcitonin (PCT), have all shown to be associated with poor outcomes in patients with SM, and have been considered for risk-stratification of children with malaria –24. Morefections , 30.Ang-2, an integral member of the Ang/Tie axis, is a promising candidate for risk stratification and triage. During normal physiological states, the Ang/Tie axis is involved in maintaining endothelial integrity through the binding of Angiopoietin-1 (Ang-1) to its receptor Tie-2. SM triggers a pro-inflammatory environment which promotes the expression and release of Ang-2, the antagonist of Ang-1, which competes for binding to Tie-2 and destabilizes the microvasculature . PreclinConducting RCTs can be costly and time-consuming and in low-and middle-income countries the challenges are even greater . To demoAn appropriate surrogate endpoint should be able to predict/measure a clinical outcome for a specific intervention and be part of the casual pathway of the disease. This is particularly true when considering biomarkers, as if they are not direct readouts of the underlying pathobiology of SM, but rather just correlated to disease outcome, they may lead to confounding findings. Moreover, biomarkers used as surrogate endpoints and the intervention being assessed should also converge on the same pathways . To dateIn contrast to lactate, the Ang/Tie2 axis has been shown to have a causal relationship to severity and death for malaria  and Ang-Identification of novel therapeutics is expensive, time consuming and risky, with many promising new chemical entities never reaching or showing efficacy in Phase III trials. In the field of cancer research, it has been estimated that de novo therapeutic development takes between 10 and 17 years with cost estimates of 1–2 billion USD . HoweverThe primary hurdle in drug repurposing is the identification of appropriate drugs to test. A multitude of databases, data mining tools and compound libraries are emerging to help the scientific community sift through the plethora of potential candidates . For exaA direct example of drug repurposing used in the context of SM is rosiglitazone , 54. Rosversus venipuncture) and matrix used , serum); fresh versus frozen samples; the platform used to detect and quantitate the marker(s) ; patient population .The future use of Ang-2 and other biomarkers in RCTs has some important limitations that need to be considered. Although these molecules are independent and quantitative markers of severity and outcome, it is unlikely that any single clinical or laboratory measurement will be uniformly predictive. Therefore, algorithms that combine predictive clinical  to risk-stratify children and adults into RCTs; through the use of validated surrogate endpoints of mortality; and, via the search for safe FDA-approved drugs that modulate these underlying causal pathways Fig. .Fig. 1Dy


Head porters constitute the mobile population who are at an increasing risk of being infected with malaria. They move around the city to carry out their duties with no accommodation. Therefore, they sleep wherever they find themselves in the evening and do not benefit from most of the malaria intervention programs such as the use of long-lasting insecticide net. The inability to identify them because they are mobile means that they can continue to drive malaria transmission even if malaria in the general population is controlled.  This study assessed the factors associated with malaria infection among head porters in the Agbogbloshie market in the Greater Accra Region of Ghana.  A total of 218 head porters were recruited from the Agbogbloshie market, and blood samples were collected from participants to test for malaria parasite infection using Rapid Diagnostic Test (RDT) and microscopy and were interviewed using a closed-ended questionnaire. The data were analyzed using Stata version 15. Simple descriptive statistics, Pearson chi-square, and Multiple Logistic Regression were performed with significance set at <0.05. Plasmodium falciparum (21/218) was the main parasite detected in all positive blood samples. Age and marital status are significant factors associated with malaria infection among head porters. Age group 40 years and above had 89% (AOR 0.11 (CI 95% = 0.01‐0.98)) reduced odds of getting malaria compared to those below 20 years, while those who are single are 3.52 times more likely to be infected with malaria compared with those who are married (AOR (95%CI) = 3.52 (1.13‐10.92)).  The study revealed 12% (CI 95% = 8.2‐16.9) and 9.6% (CI 95% = 6.3‐14.4) infection of malaria using RDT and microscopy, respectively.  This study concludes that the increasing age of head porters significantly decreased the probability of malaria infection, while head porters who are single have greater odds of being infected with malaria. Age and marital status are important factors to be considered for malaria intervention programs in head porters. The World Health Organization (WHO) report as at December 2019, indicated an estimated 228 million cases of malaria globally, out of which 405,000 deaths occurred mostly in African countries [Plasmodium falciparum malaria from 40% to 24% in the period 1900-1929 and 2010-2015, respectively [In Africa, there has been a long-term decline in the prevalence of Ghana is among the 15 countries with malaria burden in the world, accounting for 4% of the global cases and 7% of all malaria cases in West Africa , 3.Testing of suspected malaria cases in Ghana has gone up significantly from 39% to 78% from 2013 to 2016, and this has increased laboratory-confirmed cases from 143 per 1,000 population to 166 per 1,000 population, leading to a decline in malaria attributable mortality from 19% in 2010 to 4.2% in 2016 [Plasmodium species [Over the past decade, Ghana has made some progress in reducing malaria morbidity and mortality, and this resulted in decreased dominance of the various  species .Notwithstanding this achievement, there is a high prevalence of malaria in some districts in Ghana especially in the rural areas compared with the urban centers. The 2016 multiple indicator survey shows a 28% prevalence of malaria in the rural areas compared with 11% in the urban areas , 6. The The human population movement (HPM) has been described as a major challenge facing malaria control and elimination programs . Mobile Human population growth is one of the important factors in malaria transmission, and for any intervention program to be successful, this factor must be taken into consideration , 10. UrbThe study was conducted in the Agbogbloshie market in the Accra Metropolis in the Greater Accra region of Ghana. Agbogbloshie is located in the Ashiedu Keteke, a submetro of the Accra Metropolis in the Greater Accra Region. The 2010 population census report indicated that the population of Agbogbloshie area is about 8,305 with females constituting 54% while males form 46% . The marThe study population was made up of head porters who reside at the Agbogbloshie market. Most of these head porters are internal migrants who travelled from rural areas especially from the northern regions of Ghana to the capital city, Accra, to search for menial jobs. The exact total population of head porters in Accra including Agbogbloshie is unknown because they are always missed out during household or institutional surveys. However, the estimated population of head porters in Accra ranges from 2,300 to 160,000 , 15. TheN = (Z2p(1 − p))/d2 where, Z is z statistic for a 95% level of confidence, p is expected prevalence or proportion in the population based on previous studies (15.1%) [d is the margin of error or precision (0.05).A cross-sectional design was used in this study. The sample size for the study was determined using the formula   (15.1%) , and d iN = (1.962 × 0.15(1 − 0.15))/(0.052) = 195.9.Using the following formula: With the addition of the 10% nonresponse rate, the total minimum sample size for the study was 215.The outcome variable for the study is malaria infection which was tested by RDT and microscopy. Independent variables included age, sex, education, marital status, malaria awareness, causes, transmission, and preventive measures.Agbogbloshie market was selected because it is highly populated with head porters, who are also called “kayayie” in the local parlance. The National Office for the Association of head porters in Ghana is also located in the Agbogbloshie market. Information regarding the study population was taken from the President of the National head porters association in Ghana to help identify the location and time from which they can be contacted. The participants were enrolled through a simple random sampling method where the participants were asked to pick at random a piece of paper with “yes” or “no” inscriptions from a bowl. Those who chose a paper with “yes” were enrolled while those who chose “no” were not enrolled. Head porters 18 years and above were included in the study. During the enrolment, a durbar was organised by the President of the association to assemble all the head porters available on three different occasions. In the first durbar, all the head porters who were enrolled were given different unique identification codes. These first batch of participants were asked not to come again during the second and third durbars. Those who came during the second durbar and were enrolled, were also given different unique identification codes, and were asked not to participate in the subsequent durbar. This same procedure was repeated for the third time before the required sample size was obtained. Written consent was taken from the participants who were eligible for the study. Those who did not consent were excluded. A closed-ended interviewer questionnaire was used to obtain information about the factors associated with malaria transmission. The interviewer questionnaire was pretested among the head porters who reside at the Kaneshie market within the Greater Accra Metropolis.For each participant, after being interviewed, the blood sample was taken and tested for the presence of malaria parasites.Plasmodium by microscopic examination of the thick blood smears stained with Giemsa.Finger prick blood samples were collected from participants to test for malaria using the Rapid Diagnostic Test (RDT) and microscopy. The RDT malaria screening was performed using the CareStart™ Malaria test kit (Histidine Rich Protein 2) HRP2 (Pf)  following the manufacturer's instruction. This kit was recommended by the Ghana National Malaria Control Program (NMCP). Finger prick blood samples from participants were also used to make thick smears, and examined for malaria parasites after staining with 5% Giemsa. Blood smears were read by two trained microscopists using the World Health Organization  standarμl blood/#of WBC counted) × #of parasites counted.Parasite density per microliter of blood was estimated using the formula   for processing and analysis. The outcome variable is malaria infection. Two approaches of analysis were carried out, which involved descriptive and inferential statistics. Descriptive analysis was performed by running frequencies on all categorical variables and mean values for discrete variables. To assess the significant difference between the RDT and microscopy malaria prevalence, a 95% confidence interval error bar chart was adopted. Interrater agreement between the two malaria estimates performed by RDT testing and by microscopy was further assessed using the kappa statistics. Cohen's kappa statistics were calculated using the estimates of the observed and expected agreement between RDT and microscopy (two-by-two table). Inferential analysis was performed by cross-tabulations to assess the Pearson chi-square test of independence. Univariate and multivariate binary logistic regression analyses were performed separately. During the initial univariate analytical procedure, the backward selection model was adopted to select potential predictors in the study. A A total of 218 participants (over 100% response rate) were recruited at the Agbogbloshie market for the study. The age ranged between 17 years and 65 years with a mean (SD) of 29.5 (±10.32) years. The majority of the respondents were females (99.1%) with more of the head porters within the age group 20-29 years (41.7%). Also, the head porters who were married were more (51.8%), and those with no formal education were the most involved (67.9%) .The presence of malaria infection in participants using RDT and microscopy is presented in the Meanwhile, Cohen's kappa for the two tests (RDT and microscopy) was 0.21 while the observed and expected agreement was 84.9% and 80.7%, respectively.Plasmodium falciparum species. Most of the parasites density was below 200/μl (71.4%) with 19.1% having a density of 400/μl and above  for “awareness of malaria”, “causes of malaria”, “transmission of malaria,” and “preventive knowledge of malaria” of the study variables  of getting malaria by using the RDT test kit compared to head porters below the age of 20 years (AOR (95%CI) = 0.11 (0.01‐0.98)). Interestingly, head porters who are single were 3.52 times more likely to be infected with malaria compared with those who are married (AOR (95%CI) = 3.52 (1.13‐10.92)) . The HosPlasmodium falciparum was the main parasite detected in all positive blood examinations using microscopy. Most of the participants who tested positive have parasite density between 16 and 199 parasites/microliter.This study has demonstrated 12% and 9.6% of malaria infection among head porters using RDT and microscopy, respectively, and that age and marital status (being single) are the significant factors associated with this infection. Although age, marital status, awareness of malaria, causes of malaria, knowledge on malaria transmission, and malaria preventive knowledge had significant association with malaria infection, only age and marital status were observed to be statistically significantly associated with malaria infection after adjusting for study variables. The prevalence of 12% and 9.6% are relatively lower compared to what was reported in Bosomtwe-Atwima Kwawoma in Ghana a decade ago  and lowePlasmodium falciparum as the main parasite responsible for malaria in head porters [Plasmodium species accounting for 99.7% of all malaria cases in the WHO African region and causing malaria morbidity and early mortality in sub-Saharan African regions is falciparum [Plasmodium falciparum infections were missed by microscopy but were detected by the RDT. In relation to this, a previous study also identified a similar result using RDT and microscopy which detected a prevalence of 27.4% and 21.6%, respectively [Plasmodium falciparum than the RDT, and these have been explained by WHO. The difference between RDT and microscopy results could also be due to low parasitemia in the tested samples [This study corroborates with a previous study which also observed  porters . This prlciparum . Again,  samples .Age has been identified to be statistically associated with RDT malaria prevalence as indicated in this study. It has been established that age and host genetic factors are important determinants of malaria prevalence , and manThis study has also demonstrated that head porters who were not married are more likely to be infected with malaria compared with those who are married. Over 76% of the asymptomatic malaria infections were recorded among head porters who are single, and those who are single have greater odds of being infected with malaria. This finding is consistent with our previous study which reported that marital status is one of the demographic factors that affected malaria infection . MarriagThe cross-sectional nature of the design of this study precludes the generalizability of the findings to other groups of people living in malaria-endemic regions. Social desirability could also have affected the responses of some of the study participants. The findings of this study are however very important for any malaria intervention targeting head porters in the Greater Accra Region of Ghana.This study concludes that the increasing age of head porters significantly decreased the probability of malaria infection, while head porters who are single have greater odds of being infected with malaria. Age and marital status are important factors to be considered for malaria intervention programs in head porters.