Plasmodium falciparum infections. Besides PfHRP2, the PfHRP3 antigen contributes to the detection of P. falciparum infections in PfHRP2 RDTs. However, the performance HRP2-based RDT is affected by pfhrp2/3 gene deletions resulting in false-negative test results. The objective of this study was to determine the presence and prevalence of pfhrp2/3 gene deletions including the respective flanking regions among symptomatic patients in Assosa zone, Northwest Ethiopia.Rapid diagnostic tests (RDTs) targeting histidine rich protein 2(HRP2) are widely used for diagnosis of P. falciparum positive samples confirmed by quantitative PCR were included for molecular assay of pfhrp2/3 target gene.A health-facility based cross-sectional study was conducted in febrile patients seeking a malaria diagnosis in 2018. Blood samples were collected by finger-prick for microscopic examination of blood smears, malaria RDT, and molecular analysis using dried blood spots (DBS) prepared on Whatman filter paper. A total of 218 P. falciparum positive samples, exon 2 deletions were observed in 17.9% of pfhrp2 gene and in 9.2% of pfhrp3 gene. A high proportion of deletions in short segments of pfhrp2 exon1-2 (50%) was also detected while the deletions of the pfhrp3 exon1-2 gene were 4.1%. The deletions were extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene (above 30%). Of eighty-six PfHRP2 RDT negative samples, thirty-six lacked pfhrp2 exon 2. Five PfHRP2 RDT negative samples had double deletions in pfhrp2 exon 2 and pfhrp3 exon2. Of these double deletions, only two of the samples with a parasite density above 2000 parasite/µl were positive by the microscopy. Three samples with intact pfhrp3 exon2 in the pfhrp2 exon2 deleted parasite isolates were found to be positive by PfHRP2 RDT and microscopy with a parasite density above 10,000/µl.Of 218 pfhrp2/3 gene including the flanking regions. Pfhrp2/3 gene deletions results in false-negative results undoubtedly affect the current malaria control and elimination effort in the country. However, further countrywide investigations are required to determine the magnitude of pfhrp2/3 gene deletions and its consequences on routine malaria diagnosis.This study confirms the presence of deletions of  Plasmodium falciparum and Plasmodium vivax co-exist in Ethiopia [Plasmodium falciparum is responsible for most of the malaria-associated deaths in malaria endemic countries. Accurate malaria diagnostic tools and prompt treatment play an important role in reducing morbidity and mortality of malaria [Despite significant progress made in the last decade towards malaria control and elimination in most malaria-endemic countries in the world, malaria is still a major public health problem . PlasmodEthiopia . Plasmod malaria .To date, the approach to malaria diagnosis depends on the specific requirements of the malaria program and the dynamics of malaria transmission. Microscopy and rapid diagnostic tests (RDTs) are the most common front-line diagnostic tools in clinical settings. Molecular diagnostic methods are recently used in countries with low-transmission setting and/or approaching malaria elimination phase .P. falciparum and non-falciparum malaria parasites, respectively. In Ethiopia, where P. falciparum predominates, PfHRP2 RDT is widely used [P. falciparum parasites due a deletion of parts or all of the pfhrp2 gene [P. falciparum has been shown to share high homology with PfHRP2 [P. falciparum infections in PfHRP2 RDTs, but the performance of these RDTs could also be affected if there is a deletion of the pfhrp3 gene in P. falciparum [Malaria RDTs targeting histidine-rich protein-2 (HRP2) and lactate dehydrogenase (LDH) are commonly used for diagnosis of ely used . Howeverrp2 gene , affectsh PfHRP2 . This Pflciparum .pfhrp2 and pfhrp3 genes in P. falciparum populations could result in false negative RDT-based diagnosis of malaria [P. falciparum populations containing these variants of the pfhrp2 and pfhrp3 genes would be selected and would remain a major threat to existing malaria case management and control efforts [Interestingly, partial or entire deletion of  malaria , which w efforts .pfhrp2 and/or pfhrp3 genes have been reported in different geographical regions including the Peruvian Amazon Basin [pfhrp2 and pfhrp3 gene deletion on the performance of PfHRP2RDTs have also been evaluated in a few East African neighbouring countries of Ethiopa, such as Eritrea [pfhrp2/3 gene deletions in the Ethiopian P. falciparum populations. Moreover, there are no reports of pfhrp2/3 genes deletions in Assosa zone in Northwest Ethiopia. Thus, objective of this study was to assess the presence and prevalence of pfhrp2/3 genes deletion as well as the respective flanking regions among P. falciparum isolates in this area.In recent years, deletion of on Basin , Brazil on Basin , India [on Basin , and Senon Basin . The eff Eritrea , Sudan [ Eritrea , and Ken Eritrea . Therefopfhrp2/3 gene deletion in Sudan [pfhrp2/3 gene deletion on the northwestern border of Ethiopia. The geographic locations of the study sites was taken by handheld GPS  and the map was generated using ArcGIS version 10.0 software  were spotted on Whatman™ FTA™ filter paper  for molecular assay.Plasmodium parasites. Briefly, Giemsa working stain solution prepared by a stock volume of Giemsa with nine volumes of buffered water with pH 7.2. Thin blood smear fixed with methanol solution and staining with Giemsa working stain for 10 min on the staining rack. Thin smears were used to identify Plasmodium parasites species while the thick smears were examined to detect and measure the density of the parasite against 200 white blood cells (WBC), assuming mean WBC count was 8000/μL, as per WHO recommendations [Thin and thick blood smears were prepared and stained with Giemsa stain working solution diluted in 10% buffer for microcopy detection of ndations . The CarPlasmodium falciparum identification were confirmed by SYBR green based quantitative PCR (qPCR) assay by targeting the 18S rRNA gene using species-specific primers as described elsewhere [Genomic DNA was extracted from blood DBS using Chelex-Saponin method as described previously . Plasmodlsewhere . AbsolutP. falciparum positive samples with Ct-value < 37 were included for pfhrp2/3 molecular analysis to minimize false negative finding of pfhrp2/3 associated with low density infection in this study . The short segment, exon1-2, spans the end of exon1 to the start of exon2. The longest segment, exon 2, consists of the entire exon2 which is the main amino acid coding region of pfhrp2 and pfhrp3 genes. PCR amplifications were performed on upstream (PF3D7_0831900 (MAL7P1_230) and downstream (PF3D7_0831700 (MAL7P1_228) flanking region of pfhrp2 gene. Likewise, PCR amplifications were also performed on upstream PF3D7_1372100 (MAL13P1_475) and downstream (PF3D7_1372400 (MAL13P1_485) flanking regions of pfhrp3 gene. The study flow chart for molecular analysis of pfhrp2 and pfhrp3 is indicated in Additional file After confirmation of pfhrp2, pfhrp3 genes and their respective flanking regions were carried out in the total reaction mixture of 25 μl using 2X Go Taq hot start green master mix . 0.4 μM each of forward and reverse primer, 3 μl of template DNA for each pfhrp2 exon2, pfhrp3 exon2 and pfhrp3 exon1-2, 2 μl of template DNA for each pfhrp2 exon1-2, MAL7P1_230 and MAL13P1_475, 1 μl of template DNA for each MAL13P1_485 and MAL7P1_228 were used based on PCR conditions and primer sequences as described previously [pfhrp2, pfhrp3 and their respective flanking genes. Likewise, DNA of laboratory strains Dd2 (MRA-150G) and HB3 (MRA155G) were used as negative control for the PCR amplifications of pfhrp2 and pfhrp3 including their flanking genes, respectively [pfhrp2/3 PCR negative samples were repeated to confirm the absence of the target gene (pfhrp2/3 exon 2 and exon 1–2) including flanking regions.PCR amplification of eviously , 20, 21.ectively . All pfhpfhrp2, pfhrp3 and their flanking genes were separated by electrophoresis on a 2% agarose gel by staining with SYBR safe in a Tris Borate EDTA (TBE) buffer. The DNA bands were visualized in a UV transilluminator and the expected amplicons size compared to 1 kb DNA ladder. Primer sequences, PCR conditions and expected amplicon size of pfhrp2/3 genes are indicated in Additional file The amplified fragments of pfhrp2 /3 gene deletions were analysed using Statistical Package for Social Sciences (SPSS) version 20. P value < 0.05 were considered as significant.The proportion of P. falciparum positive samples by qPCR were included based on inclusion criteria for molecular analysis of pfhrp2, pfhrp3 gene and their flanking gene. Of the study participants positive for P. falciparum by qPCR, 56% (122/218) and 62.8% (137/218) of the positive cases were female and in the youngest age group (5–24 years), respectively. PfHRP2 RDT and microscopy detected approximately 60% of infections that were detectable by qPCR. There is statistical difference in positivity rate of PfHRP2 RDTs with respect to gender (P = 0.028) and age group (P = 0.05)  of pfhrp2 gene and in 9.2% (20/218) of pfhrp3 gene. Likewise, deletions of the pfhrp2 exon1-2 gene exhibited in 50% (109/218) of the isolates while deletions of the pfhrp3 exon1-2 gene were 4.1% (9/218). The deletions extended to upstream and downstream of the flanking genes of both pfhrp2 (MAL7P1_230 and MAL7P1_228) and pfhrp3 (MAL13P1.475 and MAL13P1.485) accounting for 40% of the deletions. The deletion pattern of Pfhrp2/3 and its flanking region was varied in different isolates of P. falciparum. Of the 218 P. falciparum isolates, 37.2% of the isolates did not have deletions in the pfhrp2/3 gene, including its flanking regions  of the isolates collected at the Assosa health centre (Table The pattern of deletions in pfhrp2/3 genes deletions were observed in both high and low transmission seasons. The frequency of pfhrp2 exon 2 deletion was 19.9% (30/167) in high transmission season and 17.7% (9/51) in the low transmission season. The frequency of pfhrp3 exon 2 deletion was 8.4% (14/167) and 11.8% (6/51) in high and low transmission seasons, respectively. The extent of deletions of pfhrp2/3 exon2 varied by the study site. High proportion deletion of pfhrp2 exon2 (46.2%) was observed in the Assosa district during the high transmission season followed by 28.6% of pfhrp2 exon2 deletion in Bambasi district during low transmission season  deletions observed in pfhrp3 exon 2  samples lacked 6/86 samppfhrp2/3, a high proportion of negative samples of PfHRP2 RDT lacked exon1-2 of pfhrp2  compared to exon 1–2 of pfhrp3 . Out of eighty pfhrp2 exon1-2 PCR negative samples, only five samples were tested positive for microscopy  of the clinical isolates did not have deletion in exon 2 and exon 1–2 of pfhrp2/3 region including the flanking regions.This study confirmed the presence of a considerable number of P. falciparum, microscopy and RDT were able to detect almost 60% of the infections detected by qPCR. If these large number of false negatives individual remain undetected and untreated, they could likely serve as malaria reservoirs and accelerate the onward transmission of malaria in the community[pfhrp2/3 gene in the P. falciparum populations.Of 218 positive samples for community. High prpfhrp2 exon2 deletion in this study  varied with the findings reported by previous studies such as comparable results in Kenya [pfhrp3 exon2 deletion  in this study was higher than study in Kenya [pfhrp2/3 genes deletions were observed in both high and low transmission seasons in this study area. This finding contrast with other studies in Rwanda [pfhrp2/3 genes deletions.The prevalence of in Kenya , lower rin Kenya , Mozambiin Kenya , China [in Kenya , and higin Kenya , Eriterain Kenya  and Sudain Kenya . Likewisin Kenya , but lowin Kenya , Eriterain Kenya , Ghana [in Kenya , and Honn Rwanda  and Zanzn Rwanda . The posn Rwanda , 30, hosn Rwanda , 32, drun Rwanda , geograpn Rwanda , sample n Rwanda –37 used pfhrp2 /3 exon2 and exon1-2, and upstream and downstream flanking regions of pfhrp2/3 compared to WHO pfhrp2/3 deletion threshold level [exon 2 of pfhrp2, the main amino acid coding region of the pfhrp2 gene. Of these PCR negative Pfhrp2 exon 2 samples, only three of the samples with a parasite density above 1000 / µl were microscopic positive while the rest were submicroscopic. Moreover, high prevalence of deletion was detected in short segmented of pfhrp2 exon1-2(50%) and pfhrp3 exon1-2(4.1%). High proportion of deletions (> 30%) was also extended to the downstream and upstream of the flanking regions in pfhrp2/3 gene. The complete and partial pfhrp2/3 genes deletions observed in this study would undoubtedly impact the diagnostic performance of PfHRP2 RDTs.Out of a total of 39.5% (86/218) PfHRP2 RDT negative samples in this study, a high proportion of PCR confirmed deletions were observed in ld level . Approxipfhrp2/3 gene in a certain region will be suspected if there is an increase in discordant reports between routine diagnostic tools, such as microscopy positive and PfHRP2 RDT negative [pfhrp2/3 gene deletions. Therefore, positive and negative microscopic results were included in this study to reduce underestimation of the true prevalence of pfhrp2/3 gene deletion due to submicroscopic infection. As a result, thirty-three samples with the pfhrp2 deletion gene were found to be submicroscopic in this study and included only those samples with a Ct value below 37 using qPCR (and hence had > 9 parasite density/µl) to minimize the risk of finding false deletion results associated with a single copy of the target gene (Pfhrp2) and low-density infection. P. falciparum positive clinical isolates with pfhrp2/3 gene deletion may selectively increase if there is no continuous monitoring associated with submicroscopic infections and PfHRP2 RDT negative results. Those parasites with submicroscopic parasite densities with pfhrp2/3 gene deletions could be potentially infectious to mosquitoes and contribute to ongoing malaria transmission which could in turn challenge malaria control and prevention.According to the WHO protocol, a deletion of the negative . The propfhrp2 exon2 positive by PCR. The likely cause of these false negative PfHRP2 RDT results could be due to low levels of parasitaemia that could be below the detection threshold of PfHRP2 RDT in those submicroscopic infections and absence of PfHRP2 antigen due to host immune response in the high transmission areas [In this study, 58.1% (50/86) false negative PfHRP2 RDT results were found to be on areas .pfhrp2/3 genes and their flanking regions were PfHRP2 RDT negatives in this study. The number of PCR double negative samples of pfhrp2 exon 2 and pfhrp3 exon 2 were low compared to previous studies in Peru [pfhrp2 exon 2 and pfhrp3 exon 2. Two PfHRP2 RDT negative samples were microscopic-positive with a parasite density above 2000 parasite/µl while the remaining three samples were submicroscopic. The presence of double deletions of pfhrp2/3 genes in this study would indicate the potential challenge of PfHRP2 RDT based malaria diagnosis in the study area.All PCR double negative samples in  in Peru  and Ghan in Peru . In the pfhrp3 gene to PfHRP2 RDT positive results is noted in the study under report. Even though only three pfhrp3 exon2 PCR positive samples were lacking pfhrp2 exon2, these three samples were confirmed positive by PCR and microscopy with parasite density above 10,000/µl in the absence of pfhrp2 gene suggesting the contribution of pfhrp3 exon2. This finding indicated that the structural homology between epitopes of PfHRP2 and PfHRP3 antigens allows cross-reaction of the monoclonal antibodies of PfHRP2 with PfHRP3 [pfhrp3 exon2 might underestimate the true prevalence of pfhrp2 gene deletion in this study.The contribution of intact h PfHRP3 , which ih PfHRP3 , 27. Indpfhrp2/3 gene in P. falciparum isolates in Assosa zone in Ethiopia. Previous studies have indicated that deletion of pfhrp2 in Dd2 and pfhrp3 genes in HB3 laboratory strains was resulted from deletion at distinct and unlinked positions on chromosome 8 and 13, respectively [pfhrp2/3 gene in this region may be the selective pressure of P. falciparum isolates with pfhrp2 deletion. This study is located in the Assosa zone, on the border with Sudan, which has a high transmission of malaria. Consequently, intense and routine screening of malaria with a large number of RDTs may pose selective pressure on P. falciparum and results in a considerable number of parasites carrying pfhrp2/3 gene deletion in this region. Furthermore, in the high transmission setting, polyclonal infection is common. pfhrp2/3 deleted and non-deleted strains may co-exist within an infected individual and result in positive PfHRP2 RDT [pfhrp2/3 deletion in polyclonal infections of P. falciparum using a multiplex qPCR and its effect on malaria diagnosis. Further investigations of the PfHRP2 antibodies in the plasma of P. falciparum patients infected with pfhrp2/3 deleted and non-deleted strains will clarify the impact of gene variation on the host immunogenic responses.However, it is yet unclear what causes partial or complete deletion of ectively , 41, 42.HRP2 RDT . Future pfhrp2/3 gene deletion in polyclonal infections of P. falciparum. This study was conducted in a limited geographic area.This study has certain limitations. Measurement of PfHRP2 antigen in the plasma was not performed. The molecular methods of this study could not show pfhrp2/3 gene deletions in P. falciparum isolates in the study area according to the WHO protocol [pfhrp2/3 gene could affect malaria control and elimination efforts in the country. Hence, further country-wide assessment on the magnitude of pfhrp2/3 deletions is important before considering alternative malaria diagnostic strategies.This study confirms the presence of more than 5% of protocol . The higAdditional file 1: Study flow chart for molecular analysis pfhrp2 and pfhrp3 gene.Additional file 2: Primer sequences, PCR conditions and expected amplicon sizes of Pfhrp2 and Pfhrp3.Additional file 3: Deletion pattern of Pfhrp2/3 and their respective flanking regions among 218 samples.Additional file 4: The results of PCR Pfhrp2 exon2, PfHRP2 RDTs, Microscopy with Parasite density /µl, and qPCR with parasite density/μl and Ct values among P.falciparum positive isolates.Additional file 5: Pfhrp2 exon2 negatives, pfhrp3 exon2 , PfHRP2 RDT and microscopy positive samples.Additional file 6: PfHRP2 RDT and microscopy-based results against PCR-based results of pfhrp2 and pfhrp3 their flanking regions.


E malaria vaccine is being implemented may hamper the assessment of vaccine safety and effectiveness. This study aimed to document baseline incidence rates of meningitis, malaria, mortality, and other health outcomes prior to vaccine introduction through the Malaria Vaccine Implementation Programme.The lack of background disease incidence rates in sub-Saharan countries where the RTS,S/AS01An ongoing disease surveillance study is combining prospective cohort event monitoring and hospital-based disease surveillance in three study sites in Ghana and Kenya. An interim analysis was performed on the prospective cohort in which children were enrolled in two age-groups (the 5 to 17 months or 6 to 12 weeks age-group), capturing data in the framework of routine medical practice before the introduction of the malaria vaccine. Incidence and mortality rates were computed with 95% confidential intervals (CI) using an exact method for a Poisson variable.This analysis includes 14,329 children; 7248 (50.6%) in the 6 to 12 weeks age-group and 7081 (49.4%) in the 5 to 17 months age-group. In the 5 to 17 months age-group  the meningitis, malaria, severe malaria and cerebral malaria incidences were 92 (95% CI 25–236), 47,824 , 1919 (95% CI 1461–2476) and 33 (95% CI 1–181) per 100,000 person-years, respectively. The all-cause mortality was 969 (95% CI 699–1310) per 100,000 person-years.E vaccine.Incidence estimates of multiple health outcomes are being generated to allow before-after vaccine introduction comparisons that will further characterize the benefit-risk profile of the RTS,S/AS01Trial registration: clinicaltrials.gov NCT02374450.The online version contains supplementary material available at 10.1186/s12936-021-03670-w. Plasmodium falciparum caused 99.7% of malaria cases, and children aged < 5 years were the most vulnerable age-group, accounting for 272,000 (67%) of global malaria deaths in 2018 [Despite remarkable progress in reducing the global burden of disease due to malaria, there were an estimated 228 million cases of malaria in 2018 and 405,000 malaria deaths globally, of which 380,000 occurred in the African region [E is a pre-erythrocytic P. falciparum malaria vaccine developed for routine immunization of infants and children living in malaria-endemic countries, and is the first complementary tool to be implemented under the Global Technical Strategy for Malaria, 2016–2030. RTS,S/AS01E received a positive scientific opinion from the European Medicines Agency in 2015 [E vaccination in sub-Saharan Africa (SSA), administering three doses to children aged 5 to 9 months in areas of moderate-to-high malaria transmission, with a fourth dose 15 to 18 months later [RTS,S/AS01hs later . This pihs later .E was introduced in selected areas of Ghana, Kenya, and Malawi through national immunization programmes in the framework of the WHO-commissioned Malaria Vaccine Implementation Programme (MVIP) [E and control vaccine groups in children that started vaccination at 5 to 17 months of age [E versus girls vaccinated with control vaccines not explained by differences in risk factors, causes of death, or time to death. Detailed evaluation of these events and absence of a biological plausible explanation for a causal relationship to RTS,S/AS01E suggest that these observations were likely chance findings, possibly due to a low mortality rate in girls who received control vaccines, or low rates of meningitis in the control group [In 2019, RTS,S/AS01e (MVIP) . The MVIs of age , and acrol group .E implementation [E. The end of study results will be disclosed in a separate publication.Limited or absent healthcare and disease surveillance infrastructure, and the lack of robust disease incidence data in SSA before RTS,S/AS01entation , hamper E Phase III study [Haemophilus influenzae (DTP-HepB-Hib) vaccination schedule for the 6 to 12 weeks age-group, or from 5 months of age  for the 5 to 17 months age-group  corresponding to the two age-groups evaluated in the RTS,S/AS01II study . Those aE. Case definitions are described in Additional file E administration for 5 to 17 months age group were defined for each study outcome . Training on pharmacovigilance and the diagnosis of AESI and meningitis was provided to all sites. Job aids  were also provided, and laboratory testing capacities were enhanced to support case detection and ascertainment. For each suspected case of AESI and meningitis, a protocol-specified blood sample of 5 ml was sent to an external reference laboratory for testing (second-line laboratory). For meningitis and neurological AESI, when a lumbar puncture was conducted according to routine diagnostic practice, an aliquot of cerebrospinal fluid (CSF) was sent to the second-line laboratory if a sufficient quantity was available after first-line laboratory testing.All cases of meningitis, cerebral malaria, and ambiguous cases of any other endpoint were reviewed by a panel of external experts . Cause of death was either established from the medical records when occurring at a healthcare facility, or through verbal autopsy for children who died in the community .E administration for the 5 to 17 months age-group . Study participants who reached home visit five after this date were excluded. The According-to-protocol (ATP) cohort included all children who met eligibility criteria  or after a virtual 3-dose primary vaccination schedule mimicking RTS,S/AS01Incidence rates were computed overall and by study site, and mortality rates were computed overall, by gender, and by study site using the data collected during a follow-up period of approximately 6 months after the  primary vaccination schedule. A 95% confidential interval (CI) was computed using an exact method for a Poisson variable. For the events with an at-risk period  leading to hospitalization and cause of death were coded according to the MedDRA preferred terms. Preferred terms of other AEs leading to hospitalization were grouped into medically relevant categories for the calculation of the incidence rates  primary vaccination schedule; and foot positional deformation, as defined in Additional file The study was conducted in accordance with Good Clinical Practice and Good Pharmacoepidemiology Practices, all applicable subject privacy requirements and the guiding principles of the Declaration of Helsinki. The study protocol and associated documents were approved by relevant Ethical Review Boards  in Kombewa (Kenya), 7952 (55.5%) in Kintampo , and 2158 (15.1%) in Navrongo (northern Ghana)  in the 6 to 12 weeks age-group and 7.7 months (SD 5.1) in the 5 to 17 months age-group  were hospitalized. Among the 5464 38.1%) participants who had at least one outpatient visit, 32 (0.59%) were referred to hospital. The percentage of participants diagnosed for each endpoint is provided in Table 8.1% partE primary vaccination schedule is currently administered to children aged 5 to 9 months. Therefore, the results of the ATP cohort presented below focus on the 5 to 17 months age-group. Results for the 6 to 12 weeks age-group are provided in Table In the framework of the MVIP, the RTS,S/AS01Staphylococcus aureus, and the other  incorrectly classified as meningitis due to Parvovirus before being downgraded to probable meningitis following further review by the expert panel after the interim analysis, and clarification of laboratory testing results. The incidence rate of aetiology-confirmed, probable and/or clinically suspected meningitis was 92 (95% CI 25–236) per 100,000 person-years.Five meningitis cases were initially suspected in the 5 to 17 months age-group, among which two cases were classified as aetiology-confirmed meningitis and two as clinically-suspected meningitis. The fifth case was subsequently diagnosed as encephalomyelitis Table . None ofPlasmodium falciparum was identified in 1747 cases (82%).A total of 1561 participants (22%) experienced at least one episode of malaria (2129 episodes confirmed by rapid diagnostic test and/or microscopy) during an outpatient visit or a hospitalization. Uncomplicated malaria was diagnosed in 1512 children, which corresponds to 97% of the total number of malaria events (2059 out of 2129 episodes). The remaining 3% (70 episodes in 66 children) were severe and included two cases of cerebral malaria . The malaria incidence rate was 47,824  per 100,000 person-years , malnutrition (five cases), dehydration (four cases), gastroenteritis and thermal burn (three cases each), two cases each of asphyxia and herbal toxicity, one case each of acute chest syndrome, AIDS-related complication, anemia, bronchiolitis, HIV infection, infection, acute otitis media, pulmonary tuberculosis, renal insufficiency, sepsis, skin infection, sudden infant death syndrome, and two cases of death (cause unspecified). Approximately 29% of deaths occurred during hospitalization. The all-cause mortality rate per 100,000 person-years was 969 (95% CI 699–1310), with similar rates in girls  and boys   were reported due to pneumonia (12 cases), malaria/Two AESI were diagnosed in participants in the 5 to 17 months age-group, one case of intussusception , and one case of encephalomyelitis occurring outside the at-risk period of 6 weeks . Both AESI led to hospitalization, neither was fatal.In the 5 to 17 months age-group, 392 study participants (5.5%) experienced 802 other events  that led to their hospitalization Table .Table 7IIn the 6 to 12 weeks age-group, there were five cases of abscess at the injection site after a total of 21,149 DTP-HepB-Hib doses. No cases were reported in the 5 to 17 months age-group. No foot positional deformations were reported in either group.E malaria vaccine has recently been included in the Expanded Programmes on Immunization (EPI) of selected pilot implementation areas of Ghana, Kenya and Malawi. As RTS,S/AS01E has not been introduced as part of EPI anywhere else in the world, no post-authorization surveillance data has been generated so far. The MVIP includes an important vaccine evaluation component in which GSK is conducting a comprehensive PAP in collaboration with African scientific partners. Limited background data on the incidence of diseases of interest in the framework of this PAP are available in the SSA context. The present study enrolled children not vaccinated with RTS,S/AS01E in three study sites of moderate-to-high malaria transmission in Ghana and Kenya, countries where RTS,S/AS01E is being administered in selected areas since 2019. The study was designed to document baseline incidence data prior to RTS,S/AS01E implementation to allow a before-after vaccine introduction comparison, and more broadly to help interpretation of pharmacovigilance monitoring data after vaccine implementation. Preliminary study results pertaining to the 5 to 17 months age-group are discussed here.In the context of the MVIP, the RTS,S/AS01Overall, the mortality rate per 100,000 person-years observed in the 5 to 17 months age-group was 969 (95% CI 699–1310) ranging from 265 (95% CI 32–959) to 1681 (95% CI 1041–2569) depending on the study site. Even though a formal comparison is not possible as age-groups and study period differ, those figures are not inconsistent with the INDEPTH Network mortality estimates for the period 2006 to 2012, ranging from 820 per 100,000 person-years in children aged 1 to 4 years in Navrongo, Ghana, to 7420 per 100,000 person-years in children aged 1 to 11 months in Kisumu, Kenya [Across the study sites, the incidence rate of meningitis  was 92 (95% CI 25–236) per 100,000 person-years in the 5 to 17 months age-group. Of note, antibiotic therapy was initiated one to two days before CSF sample collection for two cases, which may have affected the CSF testing results and the final case classification. Though available data on meningitis incidence in SSA are scarce and clear case definitions are often lacking, this figure is in the range of incidence rates published for Burkina Faso  [The incidence of malaria in the 5 to 17 months age-group was similar between Kintampo, Ghana and Kombewa, Kenya  but fouAmong the list of 15 defined AESI, only one case of intussusception (IS) occurred during the pre-defined risk period (14 days in the case of IS). It is not surprising to observe few AESIs at this stage given that these health outcomes are rare and that the current interim study results include a short subject follow-up period . The estimated incidence rate of IS was 144 (95% CI 4–800) per 100,000 person-years. Although the precision around this estimate is low, it is in the range of previously published data  .E administration). No foot positional deformations were reported in either group. As the study participants were around 6 weeks of age (corresponding to the 1st DTP-HepB-Hib vaccination dose) or older at time of enrolment, the choice of a congenital anomaly was not appropriate.The operational conduct of the study was assessed by two quality surveillance indicators. The rate of abscess at injection site observed for the 6 to 12 weeks age-group  is within the expected range for this vaccine  . The absThis study also generated incidence rates of more frequent conditions affecting children in SSA, such as anemia, gastroenteritis, respiratory infections, and sepsis, for which incidence estimates are often lacking , 16. To This study has been designed to detect conditions that require access to specific diagnostic tools and expertise that is not systematically available in SSA. For this reason, multiple tools have been developed and put in place to enhance case detection/ascertainment, e.g., medical trainings, job aids, telemedicine support, second-line laboratory testing of CSF/serum samples, local laboratory support, home visits, and consultation of an external expert panel. Among other aspects, those tools have been pivotal in enhancing the routine practice of CSF collection to appropriately investigate cases of suspected meningitis, and to differentiate these with cerebral malaria, combining both first and second line laboratory results and medical judgement to reach the final diagnosis. Despite the strong capacity building and subject follow-up components of this study, data collection relies on routine practice and optimal case detection and ascertainment may not have been reached due to the complex setting. Additionally, shortages of medical supplies, limited radiological and laboratory investigational capacities and/or reduced availability of medical personnel during strikes and elections are events that may have influenced case detection and ascertainment. However, this should not affect future vaccine safety and effectiveness assessment as the post-introduction study is based on the same design and is conducted in the same conditions.E PAP, generating background incidence figures around which the before-after vaccine introduction comparison will be articulated; (2) they provide baseline figures to support pharmacovigilance monitoring of the vaccine; (3) they demonstrate the technical and operational feasibility of conducting such a large scale prospective cohort study with limited pre-existing health care and surveillance infrastructures; (4) beyond the RTS,S/AS01E PAP, they provide incidence rate estimates of health outcomes for which only simulated/modelled figures are usually available. At the end of both pre- and post-vaccine introduction studies, all subjects enrolled in the prospective cohorts will have been followed up until 2 years after the fourth scheduled vaccination dose. This time-period will cover risks periods after each vaccine dose administration, as well as assessing vaccine effectiveness and impact after the primary vaccination schedule and the booster dose.These preliminary results are key for several reasons: (1) they are the backbone of the RTS,S/AS01A plain language summary of the study is provided in Fig. Additional file 1. Study age groups and home visitsAdditional file 2. Case definitionsAdditional file 3. Pre-defined at-risk periods after the  primary vaccination scheduleAdditional file 4. Eligibility criteriaAdditional file 5. Grouping of other adverse events leading to hospitalizationAdditional file 6. Quality surveillance indicatorsAdditional file 7. List of ethical review boardsAdditional file 8. Demographic characteristics at study entry by study group and study site, According-to-protocol cohortAdditional file 9. Incidence rate per 100,000 person-years of malaria cases. According-to-protocol cohort, 6 to 12 weeks age-groupAdditional file 10. All-cause mortality rate per 100,000 person-years by gender. According-to protocol-cohort, 6 to 12 weeks age-groupAdditional file 11. Incidence rate per 100,000 person-years of other adverse events leading to hospitalization. ATP, 6 to 12 weeks age-groupAdditional file 12. Incidence rate of other adverse events leading to hospitalization, by study site. ATP, 5 to 17 months age-group


The subgroup analysis of the study sites and participants was performed to explore the source(s) of heterogeneity of the outcomes. Publication bias was assessed in the case of more than 10 studies used for pooling of the same outcome. Among 114 potentially relevant studies identified from the databases, 6 eligible studies were included for qualitative and quantitative syntheses. The results showed that the pooled mean BLLs were 7.33 μg/dL in children , 4.08–10.58; I2, 98.2%), 7.94 μg/dL in children with BLL > 45 mg/dL before chelation , 7.41 μg/dL in infants , 9.20 μg/dL in children with malaria , and 36.37 μg/dL in pregnant women . The prevalence rates of malaria among participants  were 53% in children , 24% in children with BLL > 45 mg/dL before chelation , 12% in infants , and 21% in pregnant women . The subgroup analysis of countries demonstrated that the prevalence rates of malaria among participants was 17% in Benin  and 36% in Nigeria . BLL associated with decreased risk of malaria was demonstrated by two studies conducted in Benin and Nigeria, while BLL associated with increased risk of malaria was demonstrated by a study conducted in Nigeria. BLL was associated with the risk of severe malaria, involving severe neurological features and severe anemia. In conclusion, the present systematic review and meta-analysis determined the current status of the studies on BLL and risk of malaria in African countries. Further studies are needed to investigate the impact of BLL on patients with malaria to help the clinician determine the risk of severity, such as the development of neurological features or severe anemia, among patients exposed to lead.Previous epidemiological studies showed that blood lead level (BLL) was associated with malaria infection and severity. Therefore, the present study aimed to qualitatively and quantitatively synthesize the evidence on the association between BLL and risk of malaria infection and severity using the systematic review and meta-analysis approach. Potentially relevant studies were identified from three databases using a combination of search terms. The quality of the included studies was assessed using the checklist for the cross-sectional studies developed by the Joanna Briggs Institute. The qualitative synthesis of the risk or odds of malaria infection in patients with BLL was performed as the outcome of each included study could not be pooled. The pooled mean BLL and prevalence of malaria infection of the included studies was estimated using a random-effect model. The heterogeneity of the outcomes among the included studies was assessed using the Cochran Q test and I Lead is a heavy metal extensively found in paint, gasoline, electronic waste, batteries, glazed ceramics, lead-contaminated utensils, lead-contaminated water, cosmetics, traditional medicines, industrial emissions, older homes, dust, imported toys, mining and smelting industries, and spices, and there is also the industrial legacy of lead exposure and batteries ,2,3. LeaPlasmodium. Recently, eight Plasmodium species were reported to cause naturally acquired infections in humans, including Plasmodium falciparum, Plasmodium vivax, Plasmodium ovale curtisi, Plasmodium ovale wallikeri, Plasmodium knowlesi, Plasmodium brasilianum, and Plasmodium cynomolgi [Plasmodium, P. falciparum, P. knowlesi, and some P. vivax infections were known to cause severe malaria [Malaria is caused by the infection of the protozoa genus ynomolgi ,13,14. A malaria ,16. Rece malaria .Previous epidemiological studies showed that blood lead level (BLL) was associated with malaria infection ,18,19. MThe protocol of this systematic review was registered at PROSPERO (ID CRD42021260017). The reports of the systematic review complied with the Preferred Reporting Items for Systematic Reviews and Meta-analyses (PRISMA) . The seaCross-sectional studies that reported the BLL of patients with malaria were reviewed and included if eligible. The following studies were excluded: experimental studies, animal studies, reviews, systematic reviews, comments, letters to the editor, case reports, and case series.Study selections were performed by authors based on the eligibility criteria. Any disagreement on study selection between two authors was discussed for consensus. First, the potentially relevant studies were identified from three databases; then, duplicates were removed. Second, the titles and abstracts of the remaining studies were screened; then, unrelated studies were excluded. Third, the remaining studies were examined for full texts; then, unrelated studies were excluded with reasons. Finally, the eligible studies were included for further review and meta-analysis. Data of the included studies were independently extracted into the pilot standardized datasheet by two authors. The following data were extracted from each study: author, year of publication, study site, year of conducted the study, study design, number of participants, characteristics of the participants, age range and mean age, sex, research questions, mean and standard deviation of BLL, number of malaria cases, outcome, outcome parameters, diagnostic tests for malaria, and method for BLL assessment.The quality of the included studies was assessed using the checklist for cross-sectional studies developed by the Joanna Briggs Institute . The che2 statistics. The subgroup analysis of study sites and participants was performed to explore the source(s) of heterogeneity of the outcomes. Publication bias was assessed in the case of more than 10 studies used for pooling the same outcome. All analyses were performed using Stata version 14 .Data syntheses were divided into two parts. The first part was the qualitative synthesis of the evidence of the included studies, while the second part was the quantitative synthesis of the evidence in terms of available categorical or continuous data for pooling by statistical method. Qualitative synthesis of the risk or odds of malaria infection in patients with BLL was performed as the outcome of each included study could not be pooled. The pooled mean BLL and prevalence of malaria infection in the included studies were estimated using a random-effect model. The heterogeneity of the outcomes among the included studies was assessed using the Cochran Q test and IThe searches in the three databases identified 114 potentially relevant studies. After 15 duplicates were removed, the titles and abstracts of 99 studies were examined; then, 86 studies were excluded as non-related studies. The remaining 13 studies were assessed for full texts; then, seven studies were excluded with reasons: three reviews, one systematic review, one study without data on malaria and lead, one comment, and one case report. Finally, six eligible studies ,20,21,24Characteristics of the included studies are demonstrated in 2: 98.2%), 7.94 μg/dL in children with BLL > 45 mg/dL before chelation , 7.41 μg/dL in infants , 9.20 μg/dL in children with malaria , and 36.37 μg/dL in pregnant women   .2, 99.8%), 24% in children with BLL > 45 mg/dL before chelation , 12% in infants , and 21% in pregnant women  , and 36% in Nigeria  .The prevalence of malaria among participants (2381 participants) was estimated using the data from fives studies that reported 803 malaria-positive patients ,19,20,24 18–26%) . Subgrou, 99.4%) . OverallBLL and risk of malaria infection were analyzed by five studies ,19,21,24p, 0.082; correlation values r, 0.124). However, there was a higher mean parasite density in severely anemic patients with malaria (7400 parasites/μL) compared to that in those without anemia (1700 parasites/μL). They also showed a positive correlation between BLL and hemoglobin level  and a negative correlation for malaria parasite density and hemoglobin level  [BLL and risk of severe malaria were demonstrated by two studies conducted in Nigeria  and Ugan −0.231) .2, 98.2%) [The systematic review and meta-analysis showed that the pooled mean BLL among children who were investigated for malaria was approximately 7.33 μg/dL with a high heterogeneity among the two studies  ,24. The , 98.2%) , or the , 98.2%) . Among i, 98.2%) , and sou, 98.2%) . The hig, 98.2%) . 2, 99.8%) was observed. This heterogeneity might be also caused by the difference in study designs or sites. The subgroup analysis of study sites showed that the prevalence rate of malaria was high in Nigeria (36%) and lower in Benin (17%). However, heterogeneity of the prevalence existed. Therefore, the difference in the prevalence might be due to other factors, such as geographical distribution of malaria parasites in the African region. According to the WHO report in 2020, Nigeria was the country with the highest malaria burden share (27%), while the malaria burden share in Benin was only 2% [The present meta-analysis showed that the prevalence rate of malaria was highest in children (53%), while a lower prevalence rate was demonstrated in children with BLL > 45 mg/dL before chelation (24%), pregnant women (21%), and infants (12%). Among children, a high heterogeneity of the prevalence (Iin Benin %. Howevein Benin %. HoweveFor BLL and risk of malaria infection, previous studies in Benin  and NigeThe association between BLL and malaria severity was also demonstrated by two studies conducted in Nigeria  and UganThe main limitation of the present systematic review and meta-analysis was the limited number of studies that assessed the BLL and malaria; therefore, the meta-analysis on risk or odds of malaria infection influenced by BLL could not be determined. Furthermore, a high heterogeneity of the outcomes, such as pooled mean BLL and pooled prevalence of malaria infection, among participants was observed; hence, the result of the individual study or subgroup of studies should be interpreted. In conclusion, the present systematic review and meta-analysis determined the current status of the studies on BLL and risk of malaria in African countries. Moreover, the heterogeneity of the association between BLL and malaria infection was observed. Further studies are needed to investigate the impact of BLL on patients with malaria to help the clinician determine the risk of severity, such as the development of neurological features and severe anemia, in patients exposed to lead.


Plasmodium falciparum and the development factors and the consequential impact that HIV-1 has on malaria parasite transmission. Epidemiological and clinical evidence surrounding anemia, immune dysregulation, and chemotherapy as it pertains to co-infection and gametocyte transmission are reviewed. We discuss significant gaps in understanding that are often due to the biological complexities of both diseases as well as the lack of entomological data necessary to define transmission success. In particular, we highlight special epidemiological populations, such as co-infected asymptomatic gametocyte carriers, and the unique role these populations have in a future focused on malaria elimination and eradication.Malaria/HIV-1 co-infection has become a significant public health problem in the tropics where there is geographical overlap of the two diseases. It is well described that co-infection impacts clinical progression of both diseases; however, less is known about the impact of co-infection on disease transmission. Malaria transmission is dependent upon multiple critical factors, one of which is the presence and viability of the sexual-stage gametocyte. In this review, we summarize evidence surrounding gametocyte production in  Over the past several decades, malaria/HIV-1 co-infection has become a significant global public health problem in co-endemic areas of the world. The geographical overlap between both diseases . NumerouData across epidemiologic populations of interest have consistently shown HIV-1-mediated immune deficiency is associated with higher prevalence of clinical malaria and increased parasite density . A smallPlasmodium species, transmission occurs when the female Anopheles mosquito vector ingests both male and female gametocytes during a blood meal from an infected host. While the physical act of transmission seems simple, the molecular dynamics surrounding successful transmission are complex. Specific developmental and metabolic events must occur within the mammalian host before mosquito infection can occur. Similarly, intricate physiological events within the mosquito vector are necessary for onward transmission to a new mammalian host.Significant advances have been made in reducing the burden of malaria through classic vector control, case detection and treatment strategies; however, in an era focused on malaria eradication, more attention is being diverted to reduction and prevention of transmission. For all Within the mammalian host, the parasite undergoes gametocytogenesis in which the parasite differentiates between asexual (associated with symptoms) and sexual (associated with transmission) replication. Although numerous studies have expanded upon foundational work by Once sexual commitment occurs, individual developing schizonts produce either all female or all male gametocytes . The quaVery few studies have extensively and/or thoroughly included gametocyte and mosquito infectivity data. Successful transmission occurs only after complete gametocyte maturation in the host is followed by complete parasite fertilization and development in the mosquito vector. Results from Muirhead-Thompson  cautioneWhile numerous factors have the potential to influence malaria parasite transmission, immunogenetics and fluctuations in immune responses are widely studied topics. It is well described that in malaria-endemic regions, including SSA, residents are likely to have innate genetic adaptations and acquired resistance to malaria . In 2010A study in an endemic region in West Africa showed the ability to clear chloroquine-resistant parasites was most strongly associated with age, hematocrit, and ethnic background . PatientPlasmodium spp. provides a minor protective role  and non-NTDs such as HIV-1 and tuberculosis are common. Due to the intense pressure on the host immune system to control malaria parasite infection, any immune disruption from a competing infection could adversely affect clinical outcomes and gametocyte production/clearance. Amongst NTDs, polyparasitism with ive role  but moreive role . Additioive role ], fewer in vivo and epidemiological studies that seek to further explain the impact of HIV-1 on gametocytogenesis and gametocytemia, but most studies detail only indirect effects. Interesting data from a study of asymptomatic malaria among individuals who were either HIV-1 negative or positive suggests that HIV-1 positive individuals have a greater risk of carrying gametocytes than HIV-1 negative individuals , there ectivity . The P. stration . AdditioTo date, no clinical studies have looked directly at parasite transmission in HIV-1-infected patients. However, patients with progressive HIV-1 disease are known to have a number of disease-related sequelae, including immune deficiency, anemia and alterations of lipid profiles. Further, HIV-1-positive patients are commonly prescribed antiretroviral and antifolate medications. Numerous physiologic changes that occur during HIV-1 infection independently are known to affect malaria parasite transmission, some positively and some negatively. Here we present data from a variety of studies suggesting that HIV-1-infected patients are at increased risk of being reservoirs for malaria parasite transmission.A review by In vitro findings demonstrated that increased levels of cytokines (including TNF-α and IFN-γ) correlated with a lack of gametocyte infection of the mosquito vector  were negatively correlated with peak gametocyte counts and gametocyte carriage duration  biosynthesis and the resultant depletion of LysoPC leads to dramatic induction of gametocytogenesis  indicates that many asymptomatic individuals harbor gametocytes at such low densities that they are not often detected by microscopy [reviewed in ]. AssociAt this time, the prevalence of HIV-1 co-infected asymptomatics has not been fully described due to the difficulty in ascertaining asymptomatic malaria patients. Two large studies of adults in Africa showed conflicting results in overall prevalence of asymptomatic parasitemia, with HIV-1 positive patients showing significantly lower prevalence of asymptomatic parasitemia than HIV-1 negative controls. HIV-1 positive patients not using TMP-SMX prophylaxis had an increased risk of parasitemia, suggesting that antifolates reduce parasitemia . These fP. falciparum dhfr and dhps haplotypes in the same patient population, suggesting that HIV-1 co-infection could impact the spread of drug resistance  to be answered. For example, if the question is whether circulating gametocytes are intrinsically infectious, the study design may include replacing serum from a volunteer blood sample with non-immune serum to eliminate circulating host factors that could obscure intrinsic infectivity of gametocytes to exposed mosquitoes. If, on the other hand, the question is whether a volunteer is infectious based on their current clinical or treatment status, direct mosquito feeding on whole blood would be preferable to account for circulating host factors and therapeutics that could impact parasite development in the mosquito host. Given that many of the intrinsic and extrinsic host factors noted above, some of which can fluctuate on a very short timescale, can affect gametocytogenesis and gametocytemia, and that mean gametocyte circulation time in the periphery is 3.4-6.4 days , matchedPlasmodium-infected individuals remain one of the biggest threats to malaria control and eradication programs. If HIV-1 co-infected individuals have increased prevalence, frequency, duration and/or intensity of gametocytemia, they are unwittingly and unintentionally more infectious reservoirs of parasite transmission for the rest of their community. As such, these individuals represent an ideal target for additional therapies in conjunction with ART for the protection of public health and for malaria elimination and eradication.From a public health perspective, asymptomatic VAS conceived the idea for a review on the topic. AR coordinated the review, and designed and drafted all figures. AR and EF contributed to manuscript design and drafted the manuscript. All authors contributed to the article and approved the submitted version.This work was supported by NIH NIAID R01 AI104423 .The contents, views or opinions expressed in this publication or presentation are those of the author(s) and do not necessarily reflect the official policy or position of Uniformed Services University of the Health Sciences, the Department of Defense (DoD), or Departments of the Army, Navy, or Air Force. Mention of trade names, commercial products, or organizations does not imply endorsement by the U.S. Government.The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be constructed as a potential conflict of interest.


To investigate the relationship between pulse wave velocity (PWV) and retinal vessel density (VD) measured by optical coherence tomography angiography (OCTA) in patients with normal-tension glaucoma (NTG).This retrospective study included 103 patients with NTG and 109 healthy controls who underwent glaucoma examination and PWV measurements. Each group was classified into two subgroups according to a brachial-ankle PWV of 1400 cm/s. NTG was diagnosed when the maximum untreated intraocular pressure was < 21 mmHg on three repeated measurements obtained at different times in the presence of glaucomatous optic discs , peripapillary retinal nerve fiber layer defects, and glaucomatous visual field defects. Healthy controls did not have glaucomatous optic discs or visual field defects and exhibited normal retinal nerve fiber layer thickness. The interval between glaucoma examination and PWV measurements did not exceed six months. Univariate and multivariate logistic regression analyses were performed to identify factors associated with high PWV.P < 0.05). Stepwise logistic regression analysis revealed that high PWV was significantly associated with age, mean arterial pressure (MAP), and mVD in the NTG group. Meanwhile, high PWV was significantly associated with age, MAP, and low-density lipoprotein cholesterol levels in healthy controls.PWV was higher in the NTG group than in the control group, while peripapillary VD and macular VD (mVD) were lower (all High PWV is associated with decreased mVD in NTG patients, suggesting that systemic arterial stiffness might be involved in the pathogenesis of NTG. Pulse wave velocity (PWV) is among the methods for representing systemic arterial stiffness and is a useful marker for predicting future cardiovascular events.,–,,The recent introduction of optical coherence tomography angiography (OCTA) allows for the noninvasive measurement of retinal vessel density (VD) in the peripapillary and macular areas.Although both measurements may play an important role in determining vascular pathophysiology in NTG, no studies have evaluated the association between PWV and OCTA parameters in NTG. Therefore, the present study aimed to investigate the relationship between PWV and OCTA parameters and to determine which ocular parameters are associated with PWV in patients with NTG.This retrospective cross-sectional study was performed in accordance with the tenets outlined in the Declaration of Helsinki and approved by the Institutional Review Board of Yonsei University (4-2020-0592). The requirement for informed consent was waived due to the retrospective nature of the study. We reviewed the medical records of all patients treated at Severance Hospital from January 2017 to July 2020.A total of 109 healthy controls and 103 patients with NTG were included in this study. All participants underwent ophthalmologic examinations. All the examinations were carried out as usual for glaucoma patients, or patients who visited our hospital suspected of glaucoma. These included slit-lamp biomicroscopy, Goldmann applanation tonometry, gonioscopy, dilated fundus examination, measurement of best-corrected visual acuity (BCVA), and measurement of axial length (AXL) . Spectral-domain optical coherence tomography (OCT)  and standard automated perimetry  were performed to evaluate glaucomatous changes. PWV was usually measured at health check-ups, and blood tests included hemoglobin A1c (HbA1c), total cholesterol, triglycerides, high-density lipoprotein (HDL) cholesterol, low-density lipoprotein (LDL) cholesterol, estimated glomerular filtration rate (eGFR), and uric acid. The interval between glaucoma examination and PWV measurements did not exceed six months.P < 0.05 on the pattern standard deviation plot where at least one of these points had P < 0.01, or if glaucoma hemifield test was outside normal limits.Inclusion criteria were as follows: (1) age ≥ 40 years; (2) able to confirm open angle on medical records; (3) BCVA ≥ 20/40; and (4) refractive error between +3 and –6 diopters (D) spherical and ±3 D cylindrical. NTG was diagnosed when the maximum untreated IOP was < 21 mm Hg on three repeated measurements obtained at different times on separate follow-up visits and in the presence of glaucomatous optic discs , peripapillary retinal nerve fiber layer (RNFL) defect, and glaucomatous visual field (VF) defects. Glaucomatous VF defects were defined if a cluster of at least three contiguous points had Patients with poor data quality or other ocular comorbidities were excluded: (1) signal strength less than 7, images with artifacts; (2) eyes with a diagnosis of pigment dispersion glaucoma, pseudoexfoliative glaucoma, or primary angle-closure glaucoma; (3) retinal disorder ; (4) history of ocular trauma or intraocular surgery; (5) history of brain disorder ; and (6) history of optic nerve disorder .For patients with NTG, the eye with a more severe glaucoma status was chosen as the study eye. If patients had a similar glaucoma severity in both eyes, the study eye was randomly selected. For patients with healthy eyes, the study eye was also randomly selected.Brachial-ankle PWV (baPWV) was measured using volume-plethysmography . Trained technicians and physicians placed the pressure cuffs on both arms and both ankles of the patient and performed the measurement after the patient had rested for approximately 10 minutes in the supine position. The travel path from body height was extrapolated, and baPWV was computed automatically by dividing the time difference between the pulse waves that were transmitted to the brachial and ankle arteries by the travel path. The faster value of the right- and left-side baPWV was used for analysis. The device also measured blood pressure (BP) at the same time. Systolic BP (SBP) and diastolic BP (DBP) were measured, and mean arterial pressure (MAP) was calculated as follows: [SBP + (2 × DBP)]/3.,We set a baPWV of 1400 cm/s as the reference value since this is what has been identified as an independent risk factor in the Framingham score and can distinguish patients with atherosclerotic cardiovascular disease.Under pupil dilation, OCTA  images of the peripapillary and macular areas were obtained using a 6 × 6 mm scan. The optical microangiography algorithm analyzed the changes in the complex signal  and then generated en face microvascular images. In the 6 × 6 mm scan, there were 350 A-scans in each B-scan along the horizontal dimension, and 350 B-scans were repeated twice at each location. The boundaries of the superficial and deep retinal layers were determined automatically. The inner surface of the superficial retinal layer (SRL) was defined by the internal limiting membrane. The outer surface of the SRL was denoted by the outer border of inner plexiform layer. The segmentation software automatically detected the boundaries of the retinal layers from the structural OCT cross-sectional images by measuring the gradient of OCT signals to create SRL en face images. With the aid of innate software for AngioPlex OCTA, VD was defined as the total length of perfused vasculature per unit area in a region of measurement. The innate software automatically calculated the VD in the center circle with a diameter of 6 mm. It included all the vessels in the area, including large vessels. Thus, peripapillary VD (pVD) and macular VD (mVD) were calculated. All examinations were individually reviewed by two glaucoma specialists (T.L. and H.W.B.), and images with artifacts or signal strength less than 7 were excluded.t-tests for continuous parameters and chi-square tests for categorical parameters. Univariate logistic regression analysis was used to identify the factors associated with high PWV in each group. Variables with P values less than 0.2 during the univariate step were further included in a multivariate backward stepwise logistic regression model. In all analyses, P values less than 0.05 were considered to indicate statistical significance.All statistical analyses were performed using the commercially available software SPSS . Continuous variables were summarized as mean and standard deviation. Categorical variables were summarized as frequencies and percentages. Demographic and clinical data between groups were compared using independent P = 0.006) and more frequently had hypertension (P = 0.014) than healthy controls. SBP and MAP were significantly higher , while total cholesterol and LDL cholesterol were lower  in the NTG group than in the control group. PWV was also much faster in the NTG group than in the control group (P = 0.026).In total, 109 eyes of 109 healthy controls and 103 eyes of 103 patients with NTG were enrolled. P < 0.001, P = 0.002, respectively). Average RNFL thickness and average ganglion cell–inner plexiform layer (GCIPL) thickness were thinner in the NTG group than in the control group . IOP was slightly lower (P = 0.041), and spherical equivalents (SE) were more myopic (P = 0.022) in the NTG group than in the control group, although the difference was relatively small. In the NTG group, the majority of patients  were in the early stage of glaucoma, with a mean deviation better than –6 dB in the VF test. Patients in the NTG group exhibited reduced pVD and mVD when compared to healthy controls (P < 0.001), more hyperopic (P = 0.008), and more frequently had hypertension (HTN) (P < 0.001) than the low PWV group.P < 0.05). Variables with P values less than 0.2 were further enrolled in a multivariate model. Since SBP, DBP, and MAP have multicollinearity, MAP was chosen for the multivariate model. A multivariate backward stepwise logistic regression analysis revealed that age (odds ratio (OR) = 1.151, 95% confidence interval (CI) = 1.072-1.237, P < 0.001), MAP , and LDL cholesterol  were significantly associated with high PWV in healthy controls  and more frequently had HTN (P = 0.002), diabetes mellitus (DM) (P = 0.009), and chronic kidney disease (P = 0.007) than the low PWV group. SBP, DBP, MAP , and HbA1c (P = 0.022) were significantly higher, while total cholesterol (P = 0.030) and LDL cholesterol (P = 0.017) were lower in the high PWV group than in the low PWV group. In addition, the high PWV group had significantly shorter AXL (P = 0.008), more hyperopic SE (P = 0.010) and lower pVD (P = 0.036) and mVD (P < 0.001) than the low PWV group.P < 0.05). A multivariate backward stepwise logistic regression analysis revealed that age , MAP , and mVD  were significantly associated with high PWV . This must be considered when using the results of our study to inform clinical practice, as the relationship may differ in patients with more advanced NTG or other types of glaucoma, such as POAG. Studies comparing POAG with NTG may aid in clarifying the significance of PWV in glaucoma. Lastly, because of its retrospective study design, we were unable to account for all the factors that affect the measurement of PWV and OCTA. Further studies that consider all possible factors are required.In conclusion, our study demonstrates that high PWV is associated with decreased mVD in patients with NTG, indicating that systemic arterial stiffness might be involved in the pathogenesis of NTG. These results further indicate that clinical diagnosis and treatment of NTG may require consideration of systemic arterial stiffness, which can be confirmed by measuring PWV. In addition, further research regarding the role of systemic arterial stiffness in the pathogenesis of NTG is needed.


Malaria is one of the most life-threatening infectious diseases and constitutes a major health problem, especially in Africa. Although artemisinin combination therapies remain efficacious to treat malaria, the emergence of resistant parasites emphasizes the urgent need of new alternative chemotherapies. One strategy is the repurposing of existing drugs. Herein, we reviewed the antimalarial effects of marketed antibiotics, and described in detail the fast-acting antibiotics that showed activity in nanomolar concentrations. Antibiotics have been used for prophylaxis and treatment of malaria for many years and are of particular interest because they might exert a different mode of action than current antimalarials, and can be used simultaneously to treat concomitant bacterial infections. Plasmodium and is a major public health problem, mainly in Sub-Saharan Africa. Six Plasmodium species can cause malaria in humans, but only two species are clinically relevant. The two most virulent species are Plasmodium falciparum  and P. vivax . The other species, namely P. malariae, P. ovale wallikeri, P. ovale curtisi, and P. knowlesi cause only a comparable negligible burden of disease. According to the latest World Health Organization (WHO) World Malaria Report, there were 229 million cases of malaria with 407,000 deaths, of which 67% were in children under five years of age [Malaria is a vector-borne disease caused by protozoan parasites of the genus s of age .Plasmodium parasites have a complex life cycle, including morphologically distinct forms in the vertebrate and mosquito hosts [P. vivax and P. ovale can form dormant hypnozoites in the liver that can be reactivated weeks or years after the primary infection [P. falciparum malaria is caused by the ability of asexual blood-stage parasites to cytoadhere to endothelial cells in the microvasculature of several organs, eventually causing severe symptoms and death [to hosts . They reto hosts . P. vivanfection . The patnd death . In thisP. falciparum and acts against stage V gametocytes [Due to increased efforts to eliminate malaria, the number of cases and deaths dropped from 2010 to 2016, but have remained stagnant since 2016 . Clindametocytes .The historic spread of anti-malarial drug resistance and the recent appearance of parasites with a delayed clearance phenotype following artemisinin intake, and ACT treatment failures in Southeast Asia emphasize the importance of the development of new alternative chemotherapies . In addiCurrently, WHO recommends intermittent chemoprevention with sulfadoxine–pyrimethamine (SP) in sub-Saharan Africa, for certain groups, such as pregnant women and infants, and SP plus amodiaquine as seasonal malaria chemoprevention for children aged 3–59 months. However, the emergence of SP-resistant parasites, especially in east and southern Africa, jeopardizes the effectiveness of intermittent chemoprevention . DoxycycDespite the number of available antimalarial drugs, history has taught us that resistant parasites will arise and spread if drugs are in extensive use, threatening the lives of millions of people who are infected each year . One strategy to quickly and cost-effectively find new treatment alternatives is to repurpose drugs approved for the treatment of other diseases .Drug repurposing, also referred to as drug repositioning, re-profiling, redirecting, etc., is the process of finding new medical uses for existing drugs. This is a very successful strategy, and around 25% of the annual income of the pharmaceutical industry comes from repurposed drugs . The selAntibiotics are substances with antibacterial properties that mainly target cell wall, nucleic acid, or protein synthesis . Some anP. falciparum malaria [Plasmodium species [Unfortunately, many of the antibiotics have a slow onset of action against apicomplexan parasites, resulting in a so-called delayed death effect. This means that the antibiotics need two replicative cycles to exert their action against the parasites. This slow onset of action cannot only be seen in vitro, but also in vivo in monotherapy treatments of malaria, and should therefore not be considered as the main chemotherapy to treat acute  malaria ,25. The  malaria ,27. The  malaria ,29,30. T malaria ,32, the  species . This hy species . Most an species .On the other hand, some antibiotics that disrupt essential apicoplast metabolic pathways are already highly active in the first replicative cycle . AntibioFor this review, an extensive literature search was conducted to identify publications on the antimalarial activities of antibiotics that are commercially available or have already undergone clinical tests. The key studies are summarized in Plasmodium species were completely inactive. Herein, we focused on antibiotics that showed an in vitro antiplasmodial activity in nanomolar concentrations to identify antibiotic compound classes that could display a fast onset of activity.We considered antibiotics to be all naturally or synthetically produced compounds with antibacterial activity. Historically, macrolides, tetracyclines, quinolones, and lincosamides have been the most promising classes of antibiotics for antiplasmodial activities, and recently tested antibiotics confirmed this prediction. p-aminobenzoic acid and inhibits dihydropteroate synthase (DHPS), a key enzyme in the biosynthesis of folate [After the emergence of widespread resistance by the malaria parasite in endemic regions to the widely used 4-aminoquinoline chloroquine, the folate synthesis inhibitor sulfadoxine-pyrimethamine (SP) was introduced as a first line treatment in the 1990s, as a cheap and efficacious alternative . Sulfadof folate . Pyrimetf folate ,39. Bothf folate ,41,42. Af folate . SP, howf folate .Another fixed drug combination normally not used as an antimalarial targeting folate synthesis is cotrimoxazole, which consists of trimethoprim and sulfamethoxazole (1:5 ratio). Trimethoprim is a synthetic antibiotic that belongs to the diaminopyrimidine class and has a broad spectrum of activity on bacteria by inhibiting the enzyme dihydrofolate reductase. Trimethoprim was introduced in 1960s, and was frequently used in combination with sulfonamides to treat urinary tract infections . SulfamePlasmodium infections in malaria endemic areas [Daily cotrimoxazole prophylaxis of HIV-infected patients is a well-known strategy to avoid opportunistic infections , and sevic areas ,48,49. Aic areas . Years lic areas ,52. In aic areas . A systeic areas .Plasmodium parasites, leading to cross-resistance, for example, to SP. However, a recent study showed that cotrimoxazole can control malaria infections even in regions of a high prevalence of SP-resistant parasites, and there was no evidence that its use selects for mutations that confer SP resistance [Due to the extensive evidence of the potent activity of cotrimoxazole against malaria and its broad activity against many microorganisms, this drug was recommended by WHO in 2006 as a prophylactic treatment for HIV-infected children to prevent plasmodial and bacterial infections. In addition, cotrimoxazole was also indicated for HIV-exposed uninfected children from 6 weeks of age if breastfed (WHO 2016), and for persons living with HIV ,55 sincesistance . Cotrimosistance .® (sulfadoxine-pyrimethamine + amodiaquine) for seasonal preventive treatment of malaria and further formulation improvements are in development, especially adapted to the pediatric population. In addition, new formulations of only SP are in the patient confirmatory phase  [Currently, formulation improvements are ongoing, i.e., a new pediatric formulation of SP plus amodiaquine has been recently approved (SPAQ-CO™/Supyradicines) .Therefore, overall, the already widespread use of folate synthesis inhibitors limits the potential of new/additional folate synthesis inhibitors for additional applications in malaria therapy.Streptomyces aureofaciens and Streptomyces rimosus, respectively. Tetracycline antibiotics consist of a linear fused tetracyclic core to which different functional groups are attached [P. falciparum and P. vivax malaria infections [The first tetracyclines, chlortetracycline and oxytetracycline, were discovered in the late 1940s as products of attached . They caattached . Tetracyattached ,62. A weattached . The usefections ,65,66, wfections . Tetracyfections ,69, but fections .t-butylglycylamido derivative of minocycline especially designed to overcome the mechanisms of tetracycline resistance in bacteria [P. falciparum from Bangladesh [P. falciparum from the Brazilian Amazon and showed similar results with an IC50 of around 600 nM in an assay evaluating only activity after the first cycle [P. berghei infected mice at concentrations of 3.7, 11.1, 33.3, and 100 mg/kg/day given for four days [P. falciparum when analyzed over two cycles (IC50 of 14 nM) [Tigecycline, the first marketed glycylcycline is a semisynthetic 9-bacteria . Its antngladesh , and thest cycle . In vivoour days . Althougour days , showingf 14 nM) . Even thOverall tetracyclines have proven to be valuable drugs for antimalarial therapy—novel tetracyclines might even show improved and accelerated activities and should be screened for their suitability to offer improved antimalarials.Streptomyces lavendulae in 1979 [S. lavendulae (FR-32863) were highly active against Gram-positive and Gram-negative bacteria, including clinical isolates of multidrug-resistant Escherichia coli, Klebsiella pneumoniae, Enterobacter cloacaea, Pseudomonas aeruginosa, and Mycobacterium tuberculosis [Fosmidomycin is a hidroxylaminopropylphosphonic acid isolated from  in 1979 . Fosmidorculosis ,76. Of tD-xylulose 5-phosphate reductoisomerase (DOXP), a key enzyme of the methylerythritol phosphate pathway  (MEP) [P. falciparum in vitro in submicromolar concentrations after one replicative cycle [P. vinckei-infected mice when administered every 8 h for 4 days [Fosmidomycin inhibits the 1-deoxy-y) (MEP) . The firy) (MEP) . The disve cycle  and curer 4 days . This isr 4 days .A clinical trial performed in Gabon and Thailand assessed the efficacy and safety of fosmidomycin monotherapy to treat adults with uncomplicated falciparum malaria with an oral dose of 1200 mg every 8 h for 7 days . All patFosmidomycin was proposed to be developed, together with piperaquine, by Deutsche Malaria GmbH (DMG), according to the Global Portfolio of Antimalarial Medicines . In addiMacrolides are compounds that can contain one or more lactone rings with 8 to 62 atoms . The mosP. yoeli murine malaria model confirmed their potent antiplasmodial activity [Depending on the time needed to exert their effects against Plasmodia, macrolides can be grouped as either slow or fast-acting drugs. In the first group are erythromycin and its semisynthetic derivatives, while in the second group are other antibiotics such as avermectins, borrelidin, and kitasamycin . While antibiotics from the first group were poorly active against Plasmodia, some antibiotics from the second group showed a fast onset of activity. Borrelidin and kitasamycin for example, showed an IC50 in the low nanomolar range (2 and 50 nM) when tested in vitro. In vivo tests in the activity ,94. HoweStreptomyces avermitilis. Four homolog pairs containing a major and minor component of avermectins were isolated. Avermectin B and its derivatives (avermectin B1a and avermectin B1b) are the most active macrolides against endo- and ectoparasites [Mycobacterium tuberculosis in vitro [Currently, a promising compound for preventing transmission of malaria parasites is ivermectin. It is not an antibiotic in the classical sense but an antiprotozoal macrolide drug derived from avermectin B. Avermectins are 16-membered macrocyclic lactones naturally produced by arasites . Ivermecarasites ,99,100. in vitro .Loa loa endemic areas, with only rarely occurring adverse events [L. loa microfilarial load, ivermectin can cause severe encephalopathy [L. loa [The potent effect of ivermectin on nematodes is related to its selective and high-affinity binding to different kinds of chloride channels in invertebrate muscle and nerve cells. Consequently, the cell membrane permeability increases, and the chloride ions cause hyperpolarization of the cells, leading to paralysis and parasite death . In Plase events ,106. Howalopathy . In onchalopathy . Ivermec [L. loa .Anopheles spp. mosquito took a blood meal from a recently treated host, its survival was reduced [Plasmodium sporogony was impaired [In the last years, ivermectin has been extensively investigated as a potential tool to control the transmission of malaria parasites ,111,112. reduced ,114 and impaired ,116,117.impaired . Ivermecimpaired .Plasmodium stages. Evaluations showed in vitro activities of ivermectin against asexual P. falciparum stages (IC50 of around 100 nM), and gametocytes, even though only at quite high concentrations (IC50 ~ 500 nM) [P. berghei in vitro (IC50 of around 2 µM) [P. berghei infected mice after three-doses of 1–10 mg/kg [P. falciparum sporozoites did not prevent further infection [Ivermectin could have additionally an activity on its own against human  500 nM) . In addind 2 µM)  and in v10 mg/kg . Howevernfection . Other snfection ,125,126.nfection , might bRecently, the antimalarial activity of the first ivermectin hybrids  was assessed and showed that the most active derivative was threefold and tenfold more active than ivermectin against hepatic and blood-stage infections, respectively . CurrentStreptomycin spp. [Lincosamides constitute a group of antibiotics derived from the natural products lincomycin and celesticetin, which are produced by many cin spp. . Lincosacin spp. . Celesticin spp. . The antcin spp. .N-methyl-4-n-L-proline (propylhygric acid) linked by a peptide bond with the sugar 6-amino-6,8-dideoxy-1-thio-D-erythro-α-D-galactopyranoside (methylthio–lincosamide) [The structure of lincomycin consists of a trans-osamide) . Replaceosamide) . Clindamosamide) , thereafosamide) . ClindamClindamycin monotherapy cannot be recommended due to the slow onset of action, but the combination of clindamycin with quinine is recommended by WHO to treat pregnant women during the first trimester, or in combination with artesunate or quinine when an ACT is not available . LincosaP. falciparum to develop resistances to nearly all drugs in widespread clinical use and the risk of spreading of these resistant parasites, new alternatives to treat the disease are constantly needed. In particular, novel compounds for the development of non-ACT combinations are urgently needed. In this context, drug repurposing is particularly attractive tool for discovery of new treatments for malaria and other neglected tropical diseases as costs of clinical development are reduced for this inherently resource limited market.Due to the ability of Among the advantages to use antibiotics already approved, one can list the reduced costs of clinical development, their worldwide availability, and the possibility to treat different infections at the same time. Many antibiotics target the plasmodial apicoplast, an organelle that is not present in human cells and, thus, a highly selective drug activity can be expected. Furthermore, the apicoplast is a target different from common antimalarials in use and, therefore, cross-resistance should not be found. Several antibiotics have already been used/are in use for prophylaxis, or as alternative partner drugs for the treatment of malaria, and were shown to be valuable, but others deserve to be investigated further. In particular, the development of compounds with novel chemical modifications should be closely observed. As listed in this review, there are several antibiotic classes that show promising activities, with some being already highly active from the first cycle of parasite replication.The main target of many antibiotics (not ivermectin and folate synthesis inhibitors) in Plasmodia is a single organelle, the apicoplast. Even though the main target organelle is the same, some of them show a fast onset of action and others are only active starting from the second cycle. According to current knowledge, slow-acting antibiotics impair the import and processing of nuclear-encoded proteins to the apicoplast or apicoplast DNA replication, compromising the essential metabolic pathways of the organelle, but only for the progeny. On the other hand, studies on fast-acting antibiotics suggest that the direct chemical or genetic disruption of metabolic pathways causes immediate parasite death, as in the case of fosmidomycin .Although the use of antibiotics as antimalarial drugs seems to be a promising alternative, it must be considered that the uncontrolled use of antibiotics could also promote the emergence of resistant bacteria. This has been investigated for the prophylactic use of doxycycline against malaria in military personnel ,136 and