Brachybacterium conglomeratum and Brevibacterium aureum as well as a decrease in two Lactobacillus iners operational taxonomic units (OTUs). We also identified a positive independent association between HPV-16, the most common HPV subtype linked to SIL, and Brachybacterium conglomeratum. Our work indicates that HPV infection leading to SIL is primarily associated with shifts in vaginal microbiota composition, some of which may be specific to this human population.Cervical cancer is an important health concern worldwide and is one of the leading causes of death in Mexican women. Previous studies have shown changes in the female genital tract microbe community related to human papillomavirus (HPV) infection and cervical cancer; yet, this link remains unexplored in many human populations. This study evaluated the vaginal bacterial community among Mexican women with precancerous squamous intraepithelial lesions (SIL). We sequenced the V3 region of the 16S rRNA gene in cervical samples from 228 Mexican women, including 121 participants with SIL, most of which were HPV positive, and 107 healthy women without HPV infection or SIL. The presence of SIL was associated with changes in composition (beta diversity) and with a higher species richness (Chao1). A comparison of HPV-positive women with and without SIL showed that microbiota changes occurred even in the absence of SIL. Multivariate association with linear models (MaAsLin) analysis yielded independent associations between HPV infection and an increase in the relative abundance of IMPORTANCE Human papillomavirus (HPV) plays a critical role in cervical carcinogenesis but is not sufficient for cervical cancer development, indicating the involvement of other factors. The vaginal microbiota is an important factor in controlling infections caused by HPV, and, depending on its composition, it can modulate the microenvironment in vaginal mucosa against viral infections. Ethnic and sociodemographic factors influence differences in vaginal microbiome composition, which underlies the dysbiotic patterns linked to HPV infection and cervical cancer across different populations of women. Here, we provide evidence for associations between vaginal microbiota patterns and HPV infection linked to ethnic and sociodemographic factors. To our knowledge, this is the first report of the species Brevibacterium aureum and Brachybacterium conglomeratum linked to HPV infection or squamous intraepithelial lesions (SIL). Cervical cancer is one of the most common cancers and one of the leading causes of death in women worldwide , 2. Cerv4–Lactobacillus spp. Lactobacilli provide broad-spectrum protection by producing lactic acid, bacteriocins, and biosurfactants and by adhering to the mucosa that forms barriers against pathogenic infections in the vaginal microenvironment  , and 31 were diagnosed with HPV infection and high squamous intraepithelial lesion (HSIL) (women diagnosed with CIN2 or CIN3). All women were cancer free, but cases exhibited altered results in cytological, histological, and colposcopy analyses, and control, healthy women exhibited normal results.By molecular analysis, the HPV subtype frequency was highest for HPV-16, HPV-58, and HPV-18 types, with 53.72%, 15.7%, and 9.1%, respectively, observed within cases. Within women without SIL, 32.7% were positive for HPV by PCR, and the distribution of HPV subtypes was similar to that observed in cases. Most of the women in both groups did not smoke (75.21% of cases versus 71.96% of controls), had regular menstrual periods (69.42% of cases versus 68.22% of controls), and most did not have intermenstrual bleeding (85.95% of cases versus 88.79% of controls). Statistically significant differences between groups were detected in relation to an active sexual life at the time of the study (73.55% of cases versus 92.53% of controls), use of contraceptives (66.12% of cases versus 52.34% of controls), and genital hygiene, recorded by the frequency of vaginal douching (81.82% of cases versus 52.34% of controls). More details of the characteristics of each group are described in N = 228; Adonis P = 0.002). The presence of SILs was also associated with significantly higher species richness than an absence of SILs  (P = 0.041) . The presence of SILs was associated with changes in bacterial alpha and beta diversity , with no.15E−04)  as well = 0.041) , suggestP = 0.41; Adonis P = 0.11), further strengthening the link between HPV infection, rather than SILs, on the vaginal microbiota. Because of this, we further investigated the differences in vaginal microbiota explained by HPV infection. While results are similar to when data were stratified by SIL  . MaAsLin is a multivariate linear modeling tool with boosting that tests for associations between specific microbial taxa and continuous and/or Boolean metadata. This method reduces the total amount of correlations to be tested, therefore allowing for improvements in the robustness of the additive general linear models. With MaAsLin, we found significant independent associations between HPV-positive status and an increase in the relative abundance of s (OTUs) . This ins (OTUs) . For exanfection .Several factors are known to play a role in cervical carcinogenesis, with HPV infection being one of the most important in the development of the disease . There aOur study also revealed several other factors associated with SIL status, some of which reaffirm previously reported links . FactorsAcinetobacter lwoffii, which has been previously reported in HPV-positive women  secretion, causing proinflammatory activity in the cervix, which may influence the progression of cervical intraepithelial neoplasia  and not SILs, indicating that infection itself may lead to changes in the vaginal microbial community. Among the predominant components of a healthy vaginal microbiome is the presence of  gasseri , 42, whil cancer  and withl cancer , 44. In obacilli –47. For eoplasia . In otheeoplasia .Brevibacterium aureum and Brachybacterium conglomeratum (increased in cases). While these differences were very significant, these species were not uniformly present among either group  and attending the Instituto Mexicano del Seguro Social (IMSS) in Mexico City were invited to participate as volunteers in this study. Written informed consent was obtained from all volunteers after providing detailed information about the study and its characteristics. The clinical research protocol and letter of informed consent were evaluated and approved by the Comité Local de Investigación y de Bioética de la División de Educación e Investigación Médica de la Unidad de Alta Especialidad Médica Pediatría del IMSS. All participants completed a study questionnaire that was used to obtain sociodemographic and risk factor information. Data were registered in a secured database for subsequent statistical analysis.A total of 228 Mexican women over 21 years of age who attended the IMSS from December 2003 to July 2006 were enrolled in the study. These women were divided in two groups. The first group (controls) consisted of 107 healthy women with a mean age of 42.8 years (±7.9) with three previous Papanicolaou (Pap) tests negative for HPV infection for three consecutive years (a fourth negative Pap result occurred at the time participants were invited to join the study) and diagnosed without SIL with normal cytology and colposcopy results by the treating gynecologist. The second group (cases) consisted of 121 patients with a mean age of 37.3 years (±10.9) with different degrees of SILs and were positive for HPV infection based on cytology, histology, and colposcopy examination. This group included women diagnosed with cervical intraepithelial neoplasia from 1 to 3  according to the Bethesda classification . ParticiBesides SIL and HPV status, the following study variables were included in the analysis: age, HPV subtype, smoking (daily number of cigarettes consumed at time of study), regularity of menstrual period, intermenstrual bleeding, current sexual activity status, use and type of contraceptives , vaginal douching, age of sexual debut, number of sexual partners, number of pregnancies, and number of births and miscarriages.Samples of vaginal exudate were taken by swabbing the mucosa using sterile Teflon swabs that were placed in a sterile 15-ml conical plastic tube with sterile 0.9% sodium chloride . The sample was kept at −20°C until its use for microbiome sequencing analysis.Cervical DNA was extracted directly from the brushing done by the gynecologist before the application of acetic acid for the colposcopy study. The brush was stored in 1 ml of saline solution at 4°C for transport. DNA was extracted immediately from a first centrifugation at 8,000 rpm for 5 min, and the pellet was resuspended in 200 μl of extraction buffer (SDS 1%) and proteinase K (200 μg/ml), making a modification in the digestion of the sample (53°C for 5 h) and inactivating the reaction by boiling for 5 min . Once th53–From vaginal DNA samples, the 16S rRNA gene was amplified by PCR in triplicate using bar-coded primer pairs flanking the V3 region as previously described . Each 50For 16S rRNA gene sequencing, each PCR pool was analyzed on an Agilent Bioanalyzer using a high-sensitivity double-stranded DNA (dsDNA) assay to determine approximate library fragment size and verify library integrity. Pooled library concentrations were determined using the TruSeq DNA sample preparation kit, version 2 (Illumina). Library pools were diluted to 4 nM and denatured into single strands using fresh 0.2 N NaOH. The final library loading concentration was 8 pM, with an additional PhiX spike-in of 20%. Sequencing was performed using a Hi-Seq 2000 bidirectional Illumina sequencing and cluster kit, version 4 . PCR products were visualized on E-gels, quantified using an Invitrogen Qubit with PicoGreen, and pooled at equal concentrations, according to a previous report .N of 228. Further filtering was applied to remove mitochondrion and chloroplast sequences and OTUs present in fewer than three samples. The OTU table was included as a supplemental data set in the supplemental material.All sequences were processed using mothur v.1.43.0 according to the standard operating procedure as previously described . Qualityt tests (parametric) were used based on the D´Agostino-Pearson normality test. We assessed vaginal microbial diversity and the relative abundance of bacterial taxa using Phyloseq  and a 95% confidence interval (CI). For numerical variables, Mann-Whitney (nonparametric) or Student Phyloseq  along wiPhyloseq  were usePRJNA766648).Sequences have been deposited to BioProject (accession number


Cancer of unknown primary (CUP) is a heterogeneous malignancy in which the primary site of the tumor cannot be identified through standard work-up. The survival outcome of CUP is generally poor, and there is no consensus for treatment. Here, we comprehensively analyzed the real-world data of 218 patients with CUP . Next-generation sequencing was conducted in 22 (10%) patients, one of whom showed level 1 genetic alteration. Most (60.3%) patients were treated with empirical cytotoxic chemotherapy, and two patients received targeted therapy based on the NGS results. The median OS was 8.3 months , and the median progression-free survival of patients treated with chemotherapy was 4.4 months (95% CI 3.4–5.3). In multivariate Cox regression analysis, Eastern Cooperative Oncology Group performance status (ECOG PS) of 0 or 1 and localized disease were significantly associated with favorable survival outcomes. Collectively, we found that CUP patients had a poor prognosis after standard treatment, and those with localized disease who received local treatment and those with better PS treated with multiple lines of chemotherapy had better survival outcomes. Targeted therapies based on NGS results are expected to improve survival outcomes. CUP historically accounted for 3–5% of all malignancies, but this number has recently decreased to 1–2% owing to advances in diagnostic methods including molecular profiling2. Most patients with CUP have been included in unfavorable subsets and showed poor prognosis, with a median overall survival (OS) of 6 months1. Because of the heterogeneity of CUP, there has been no consensus on the standard treatment, and empirical cytotoxic chemotherapy and palliative radiotherapy (RT) based on suspected primary sites have been generally used for patients with CUP3.Cancer of unknown primary (CUP) is a heterogeneous malignant condition for which the primary site of origin has not been identified by general diagnostic evaluation7. Moreover, the number of studies on site-specific therapy and targeted therapy based on gene expression profiling and NGS results in CUP patients has been increasing11. Some prospective studies have demonstrated better survival outcomes after tailored therapy than after empirical cytotoxic chemotherapy9, but randomized trials did not report such differences11. This suggests that there is some discrepancy between studies in terms of the efficacy of the novel targeted therapies for CUP12, which is likely due to the incomplete understanding of the characteristics of CUP.Recently, gene expression profiling and next-generation sequencing (NGS) have gained attention as tools for identifying the primary site and studying the molecular features of CUPDespite recent advances in diagnostic techniques and treatment strategies, the characteristics of CUP and treatment patterns in real-world settings are not well-known. The clinical utility of tailored therapy is also yet to be established as there are few studies on how novel targeted therapies are being applied in clinical practice. For this reason, we conducted a retrospective study on patients who were diagnosed with CUP and underwent treatment at Asan Medical Center, a 2700-bed tertiary center in Seoul, South Korea, to examine the clinical and molecular characteristics, treatment patterns, survival outcomes, and efficacy of NGS and targeted therapy in patients with CUP in a real-world setting.Between January 2009 and December 2019, a total of 218 patients with CUP were registered in the Asan Medical Center cancer registry. During diagnostic work-up, all patients underwent medical history taking, physical examination, baseline blood and biochemistry analyses, imaging studies including chest and abdomen-pelvis computed tomography (CT), and biopsy. Additionally, 94% (n = 206) of patients underwent positron emission tomography (PET)/CT as a part of the diagnostic work-up. Table Twenty-two 10.1%) patients underwent NGS, and the most common tissues used for NGS were lymph nodes (59%), bone (18%), and lung (9%). A total of 600 alterations were identified in 268 genes , and all patients showed at least one or more gene alterations. Figure 0.1% patiAmong the 218 study patients, 132 (60.7%) received first-line chemotherapy (Table The most common regimen used as second-line chemotherapy was PC (n = 12), followed by GP , CAV , and FP (n = 7). The best response to second-line chemotherapy was PR (8.5%), and 42.8% of patients showed PD. The median PFS after second-line chemotherapy was 2.1 months (95% CI 1.9–4.0). The details of third-line and fourth-line chemotherapy regimens are summarized in Supplementary Table A total of 58 (26.6%) patients did not receive treatment in our center. Among them, 20 were transferred to other hospitals after diagnosis due to the patient’s choice, 11 refused treatment, 23 were unable to receive treatment due to poor PS, and 4 were lost to follow-up after the initial diagnostic work-up.Targeted therapy was provided to two patients based on the NGS results. A patient who had NTRK fusion initially presented with abdominal pain, and CT scan showed enlarged, multiple abdominal lymph nodes in the para-aortic, aortocaval, and small-bowel mesentery areas. The pathology of the abdominal lymph node was confirmed as metastatic adenocarcinoma, and the patient was diagnosed with CUP because the primary site could not be determined through standard diagnostic evaluation. The patient was initially treated with conventional cytotoxic chemotherapy (gemcitabine-cisplatin); however, a new metastatic lung nodule was documented after 3 months (five cycles), and was thus treated with entrectinib, an FDA approved targeted therapy for solid tumor with NTRK fusion, as second-line therapy. The patient maintained a stable disease status for 9 months while taking entrectinib, but the disease had progressed as enlarged abdominal lymph nodes. As of this writing, the patient is currently participating in a clinical trial of an immune checkpoint inhibitor  as third-line therapy.The other patient who received targeted therapy had an AKT2 gain mutation and was treated with ipatasertib, an FDA-approved targeted therapy against AKT. The patient was diagnosed with CUP with involvement of the liver, lung, right ureter, and multiple lymph nodes  and was initially treated with conventional chemotherapy of PC and GP; however, the patient showed progression despite chemotherapy and was started on ipatasertib, but died after 2 weeks of treatment due to liver failure.Immunotherapy was provided to three patients. One patient with inguinal area lymphadenopathy and squamous cell carcinoma was treated with PC as first-line chemotherapy. After six cycles of PC, he remained in the SD status for 6 months during the drug holiday. The disease progressed afterward and pembrolizumab was administered as second-line chemotherapy considering the microsatellite instability (MSI)-high status observed in immunohistochemistry. The PFS was approximately 6 months during pembrolizumab therapy, and FP and re-do PC were administered as third- and fourth-line chemotherapy regimens, respectively. The patient died due to septic shock and pneumonia.Another patient treated with immunotherapy had anterior mediastinal mass, and biopsy showed poorly differentiated adenocarcinoma. Despite treatment with VIP , AP , IP , and palliative radiation therapy, the disease progressed. Pembrolizumab was administered once as fourth-line chemotherapy, but the patient died within 2 weeks before the scheduled disease evaluation.The last case is the patient who was treated with entrectinib described above in the Sect. “Treatment patterns: targeted therapy” paragraph. Currently, the patient is participating in a clinical trial of spartalizumab (anti-PD1 Ab).Forty-six patients underwent surgery . Surgeries for palliative purposes included the following: decompression operation due to spinal bone metastasis, bowel resection due to obstruction, internal fixation of humerus due to pathologic fracture related to bone metastasis, abdominal tumor mass excision for pain control, and brain tumor resection.A total of 66 patients received RT. Among them, 19 received definite RT, of whom 10 underwent concurrent chemoradiotherapy (CCRT), and the rest (n = 47) received palliative RT for pain control. The diagnosis of the ten patients treated with CCRT included squamous cell carcinoma in the head and neck lesion (n = 5), squamous cell carcinoma at the mediastinum (n = 1), squamous cell carcinoma in the lymph node on the inguinal lesion (n = 1), and poorly differentiated carcinoma at the cervical lymph node region (n = 3). The median OS of the patients treated with CCRT was 51.7 months (95% CI 40.4–not reached [NR]).The median OS of the study patients as a whole was 8.3 months (95% CI 6.2–11.4)  and localized disease  were significantly related to better OS, whereas old age (> 60) (P = 0.45) and male sex (P = 0.96) were not significantly associated with survival outcomes. Multivariate analysis also showed that ECOG PS  and localized disease  were significantly related to survival outcomes . This is likely because most of such patients were favorable subsets of CUP such as squamous cell carcinoma involving cervical lymph nodes and inguinal adenopathy13. They also found that the number of the metastatic organ systems was significantly related to survival outcomes whereas age and sex did not show such relations with survival. These results are consistent with our findings in that PS and disease extent are important factors for prognosis prediction in CUP. However, the study by Löffler et al. was different from our research in that it only included patients with adenocarcinoma or poorly differentiated carcinoma. Interestingly, Löffler et al. reported a median overall survival of 16.5 months, which is a better survival outcome than those in previous publications14 and ours. Considering that localized disease status was significantly associated with better survival outcomes in our study, such a difference in the OS results was possibly due to the differences in the proportion of patients with single organ involvement (49% vs. 15%).Some retrospective studies have been carried out on the treatment patterns and outcomes of CUP. Löffler et al. analyzed the clinical characteristics, treatment patterns, and survival outcomes of 223 patients in Germany with a CUP of adenocarcinoma or un-differentiated carcinomas, and reported that the most commonly involved organ system was the lymph node, liver, bone, and lung15. They showed recent trends in the diagnostic work-up and treatment strategy in real-world settings and presented the patient characteristics, use of diagnostic work-up, and survival outcome. Notably, a considerable proportion of 99 (2.2%) patients received targeted therapy. The OS of all patients was poor at a median OS of 1.2 months, and only 20.3% of patients were confirmed to be alive after 6 months; such poor survival outcome may have been due to the relatively old age of patients and the low proportion of properly treated patients. In contrast, our study showed a better overall survival of 8.2 months and a higher proportion of patients received anticancer treatment. Even with recent advances in diagnostic methods and treatment strategies, the prognosis of CUP is still poor as shown in our study. One of the limitations of the SEER-M-based study was the exclusion of patients aged under 66 years.Another large-scale study that included 4,562 patients using American Surveillance, Epidemiology, and End Results-Medicare (SEER-M) linked database was recently published17. However, the clinical benefits of gene expression profiling are yet to be clearly demonstrated11, and the method is not routinely recommended for diagnostic evaluation in patients with CUP18.To improve the diagnostic accuracy for CUP, new approaches are being investigated. As an example, gene expression profiling was developed to determine the primary site of the CUP, and the results revealed excellent diagnostic benefits in tumor classification with an accuracy of 85%, which is comparable to that of immunohistochemistry22. Such a wide range of the proportion of patients with an actionable gene mutation may be due to differences in NGS assays, gene panels, and the definition of actionable mutation in each study. In our study, we used the OncoKB data for classification23, and 10 (43.5%) out of 23 patients showed level 1, 2, or 3 alterations.NGS is widely used nowadays to identify actionable gene mutations in patients with CUP. In previous studies using NGS, the proportion of actionable gene mutations in CUP patients ranged from 30 to 85%20. In 2017, Varghese et al. reported the outcome of targeted therapy in patients with CUP based on the NGS results; of the 150 patients who underwent NGS, 45 showed clinical genomic alteration, and 10% (n = 15) received targeted therapy and showed varying treatment outcomes 5. In a study conducted in South Korea, 17 among 21 patients who underwent NGS showed possible clinical genomic alterations and only one received targeted therapy24. More recently, a phase 2 trial of site-specific therapy based on NGS results was conducted in 97 patients with CUP; the study showed that the 1-year survival probability was 53% and the median OS was 13.7 months, thus suggesting the possible clinical applicability of tailored therapy25. The CUPISCO study (NCT03498521) is currently ongoing, which is a randomized trial comparing individualized targeted treatment or immunotherapy with standard platinum-based chemotherapy in patients with CUP; the results of the CUPISCO study are expected to be released within a few years.With advances in diagnostic methods such as NGS, new treatment strategies are also being suggested. Some studies conducted NGS in CUP patients and suggested the possibility of personalized therapy based on NGS resultsIn South Korea, NGS for patients with malignancies has been approved and reimbursed by the National Health Insurance Service since March 2017. In our study, 23 patients underwent NGS, most of whom were diagnosed after 2017. Of note, in our cohort, targeted therapy based on NGS results was provided to two patients and showed varying survival outcomes. While one patient died despite 2 weeks of treatment with ipatasertib, another patient treated with entrectinib had a better survival outcome than that of patients treated with standard empirical chemotherapy. The reason for such different clinical outcomes is likely the small number of patients who received targeted therapy, and further large-scale prospective studies are warranted to determine the role of targeted therapy.This study has several limitations. First, the NGS panel that was used in our institution was a targeted panel sequencing for solid tumors that identifies genomic alterations in approximately 300 cancer-related genes. As such, some gene alterations that were not included in our panel would not have been identified. This may be a reason for the low proportion of patients who were treated with targeted therapy, and it may have affected the clinical outcomes in our cohort.Also, our NGS results could not suggest proper treatments for most of the patients in our cohort. We believe that this reflects the difficulties in real-world practice that optimal targetable gene alterations are hard to identify, even with NGS assays, due to the presence of multiple concurrent gene mutations in CUP patients and the limitation of the NGS assay described above. Furthermore, even if we found actionable gene mutation using NGS assay, the targeted agents for most of the actionable mutations are not readily available for clinical use, and this also may be related the unsatisfactory outcomes in patients with CUP. In order to improve the clinical outcomes of CUP patients, further investigations are needed to develop the proper method for evaluating targetable genetic alterations and to develop optimal targeted therapies.Second, as this study was conducted at a single center, there may have been selection bias and the results may have limited generalizability. The number of study patients was small, but it is a relatively large-scale study considering the rare prevalence of the CUP, and there has not been a study of this scale in Asian patients with CUP. Moreover, despite its retrospective nature, this study is meaningful because a prospective study cannot be easily conducted due to the nature of the disease and our results reflect the current trends and outcome in a real-world setting. Another limitation is that we could not systematically evaluate the adverse effects in each patient due to the heterogeneity of the chemotherapy regimen.Patients who were diagnosed with CUP and registered in our institution’s cancer registry between January 2009 and December 2019 were identified and included in the study. CUP was defined according to the initial ICD code of “unknown primary site” (ICD-0-3 code 80.9). Among them, after a comprehensive chart review, we excluded patients whose primary site was later identified through further imaging studies and/or histologic diagnosis with additional immunohistochemical staining. We reviewed the medical records of all included patients to collect data on demographic and clinical characteristics including age, sex, ECOG-PS, histopathological diagnosis, number and location of metastases, disease extent, treatment strategy, chemotherapy regimen, response to chemotherapy, and survival outcome. Disease extent was classified into localized disease  and disseminated disease. All non-localized cases were classified as disseminated diseases.26. VarDict was used to conduct somatic variant calling for single nucleotide variants and short indels27. CNVkit was used to perform Copy Number analyses28. Using common germline variants database , common and germline variants candidates from somatic variants were extracted. We identified patients who underwent NGS and analyzed their NGS results. The clinical actionability of specific molecular alterations was assessed using OncoKB, a precision oncology knowledge database (http://oncokb.org)23. According to the OncoKB database, actionable gene alterations were classified from levels 1 to 4: level 1 alterations are defined as Food and Drug Administration (FDA)-recognized biomarkers for FDA-approved drugs in a specific cancer, level 2 alterations are defined as standard biomarker recommended by the standard guidelines for an FDA-approved drug, level 3 alterations are defined as biomarker supported by clinical evidence as being predicted of response to a certain drug, and level 4 alterations are defined biomarkers supported by biological and preclinical evidence23.Genomic DNA was extracted from previously archived tumor tissues. Targeted sequencing was carried out using the MiSeq platform  with an in-house panel designed at Asan Medical Center  using the SureDesign  with the GRCh37 reference version. The Oncopanel AMC version 3 and 4 (OP AMC v3 and v4) identifies genomic alterations in 383 and 323 cancer-related genes, respectively; specifically, the OP AMC v3 examines 199 genes for SNV/INDEL, and copy number variation (CNV), 8 genes for rearrangement, and 184 hotspots, and the OP AMC v4 examines 225 genes for SNV/INDEL and CNV, 6 genes for rearrangement, and 99 hotspots. The included gene lists are provided in Supplementary Table 32.OS was calculated from the date of pathological diagnosis to the date of death due to any cause. Progression-free survival (PFS) was calculated from the date of chemotherapy initiation to the date of documented progression or death. If a patient was alive on the date of the last outpatient visit and lost to follow-up, we censored the patient at the date of the last outpatient visit date. The Kaplan–Meier method was used to calculate the OS and PFS, and the log-rank method was used to compare the OS and PFS between subgroups. Multivariate Cox regression analysis was performed to determine the prognostic value of variables. P values < 0.05 were considered statistically significant. All statistical analyses were conducted using R statistical software, version 3.6.3 This retrospective study was performed in accordance with the Declaration of Helsinki, and the protocol was reviewed and approved by the Institutional Review Board (IRB) of Asan Medical Center . Written informed consent was obtained from study participants who underwent NGS, and the requirement of obtaining informed consent from patients who did not conduct NGS was waived by IRBs because of the retrospective nature of this study.Supplementary Information.


Glaucoma is a leading cause of permanent vision loss and current drugs do not halt disease progression. Thus, new therapies targeting different drug targets with novel mechanisms of action are urgently needed. Previously, we identified CACNA2D1 as a novel modulator of intraocular pressure (IOP) and demonstrated that a topically applied CACNA2D1 antagonist—pregabalin (PRG)—lowered IOP in a dose-dependent manner. To further validate this novel IOP modulator as a drug target for IOP-lowering pharmaceutics, a homology model of CACNA2D1 was built and docked against the NCI library, which is one of the world’s largest and most diverse compound libraries of natural products. Acivicin and zoledronic acid were identified using this method and together with PRG were tested for their plausible IOP-lowering effect on Dutch belted rabbits. Although they have inferior potency to PRG, both of the other compounds lower IOP, which in turn validates CACNA2D1 as a valuable drug target in treating glaucoma. Glaucoma is a group of eye diseases that can slowly and asymptomatically steal human sight. Most glaucomatous patients do not recognize that they may have glaucoma until they suffer significant vision loss. Vision field loss associated with glaucoma occurs due to optic nerve damage that results mainly from the persistent pressure exerted by elevated IOP, especially in case of primary open angle glaucoma (POAG). POAG accounts for more than 90% of glaucoma cases all over the world and is considered to be one of the leading causes of irreversible blindness, especially in elderly people . BecauseIn drug discovery, molecular docking is a commonly used method to predict the binding mode and affinity of a ligand to a protein with a known structure. Typically, the ligand will be tried to dock against a specific region of the protein. The best binding pose will then be determined, and a docking score will be calculated typically based on the energy reduction of the two entities before and after the docking. In this situation, a lower docking score indicates a higher energy reduction and thus a tighter binding between the ligand and the protein . When thIn the current study, to further validate our target protein—CACNA2D1—as a potential target for the treatment of glaucoma, we used the docking method to identify several compounds that are structurally similar to PRG with a good affinity to the target protein. These compounds were also tested in vivo to validate their therapeutic effect.To screen for the hits of CACNA2D1, a homology model was built and five possible binding sites were identified . PRG wasStreptomyces sviceus, which has an antitumor activity [®, Aclasta® and Zometa®) [As previously reported, PRG is a promising IOP-lowering medication that targets CACNA2D1 ,10. We pactivity . ZoledroZometa®) .w/v Carbopol 981 viscous eye drops were selected as the vehicle for all the tested molecules, including PRG. Carbopol 981 is characterized by its reasonable viscosity, biocompatibility and bioadhesiveness [Because these drug molecules that we identified are soluble in an aqueous vehicle, they can rapidly drain from the eye surface upon topical application into the eye. For this reason, 0.2% siveness ,16. Carbsiveness . Bioadhesiveness ,18.The measured pH of the tested viscous eye drops  ranged bThe abilities of these molecules to lower the IOP were tested on Dutch belted rabbits and compared to the IOP-lowering effect of PRG in the same animal model. The IOP-lowering results of all the tested molecules are plotted in Regarding the safety of these new molecules as potential IOP-lowering therapies, neither PRG nor acivicin show any signs of irritation, toxicity or allergic reactions . In contPRG (≥97% purity) and triethanolamine were purchased from Sigma Aldrich . Carbopol 981 was obtained as a gift sample from Lubrizol Advanced Materials, Inc. . Acivicin (NSC163501) was purchased from Cayman Chemical . Zoledronic acid (NSC721517) was purchased from AK Scientific Inc. .Dutch belted rabbits, mixed males and females, aged 5–7 months, weighing 1.5−2.5 kg, purchased from Covance Inc. , were used to test the IOP-lowering effects of the tested molecules. All animals were examined before the study and appeared free of any clinically observable abnormalities. All rabbit eyes were healthy with no injury or history of injury. The IOP difference between the two eyes of the same rabbit did not exceed 1 mmHg. Through the whole study, rabbits had free and continuous access to food and water. All procedures including rabbits were previously approved by the Animal Care and Use review board of the University of Tennessee Health Science Center (UTHSC), Memphis, TN. The protocol also followed the Association of Research in Vision and Ophthalmology (ARVO) Statement for the Use of Animals in Ophthalmic and Vision Research and the guidelines for laboratory animal experiments .Because no CACNA2D1 crystal structure has been published to date, we built a homology model and used it for the docking studies based on the procedure described as follows: The protein sequence of CACNA2D1 was obtained from the UniProt database and was subsequently used for the online BLAST search. The voltage-gated calcium channel Ca(v)1.1 (PDB: 5GJV ) with 91The binding sites of the abovementioned homology model of CACNA2D1 were identified using SiteMap and the top five sites were used for docking studies. The structure of PRG was first prepared by Ligprep with the OPLS3e force field and was subsequently used to dock against the five sites. Similarly, ligands from National Cancer Institute (NCI) database were also prepared and docked against the same 5 sites.w/v was soaked in Milli-Q water overnight until full swelling. PRG and acivicin were dissolved separately in Milli-Q water, while zoledronic acid was dissolved in 1% w/v triethanolamine solution, at a concentration of 1.2% w/v. Equal volumes of 0.4% w/v Carbopol 981 gel and drug aqueous solutions were mixed together using a vortex mixer to produce the final bioadhesive viscous eye drops, which were kept protected from light in closed air-tight containers at 5 °C until the time of use. After mixing, the final products were composed of 0.6% w/v of each drug in 0.2% w/v Carbopol 981 gel. To ensure sterility of the final products, all the used tools and water were sterile and all procedures were performed under aseptic conditions.Because all the used drug molecules are soluble in aqueous media, they could be easily prepared in the form of aqueous solutions. Unfortunately, these aqueous solutions could be rapidly drained from the eye surface before exerting any pharmacological response. For this reason, we incorporated these molecules in bioadhesive viscous Carbopol 981 eye drops. Carbopol 981 in a concentration of 0.4% The pH of the prepared eye drops was measured according to our previously published protocol . A gram 1.Efficacy evaluationn = 3) according to our previously published protocols [w/v Carbopol gel vehicle) and used as a control. A Tono-pen AVIA  was used to measure the IOP. The baseline IOP was measured immediately before application of the eye drops and then was taken hourly until it returned back to its baseline value. At each time point, three readings were taken and averaged for each eye. The IOP measurement was repeated until we measured a fixed value at each reading. The relative efficacy of each drug molecule was evaluated by comparing the calculated pharmacodynamic parameters of the three eye drops containing the different drug molecule. The calculated pharmacodynamic parameters include maximum percent IOP reduction; the time required to reach the maximum decrease in percent IOP (Tmax); the time required for IOP to return back to its baseline ; and the total area under the percent IOP reduction-versus-time curve (AUC). All pharmacodynamic calculations were carried out using GraphPad Prism-9 software . All results were expressed as the mean ± SEM. The IOP-lowering effect of the different eye drops containing different drug molecules was evaluated using Dutch belted rabbits  according to our previously published protocols [w/v Carbopol gel vehicle) and used as a control. All eyes were visually evaluated for the appearance of irritation, toxicity or allergic reactions, such as tearing, inflammation, corneal swelling, conjunctival redness, hyperemia or swelling, etc., after 1, 2, 3, 4, 6, 8, 24, 48 and 72 h after the application. In addition to the visual evaluation, slit-lamp examinations were performed after 1, 2, 3 and 7 days. More examinations were performed to evaluate any irritation, toxicity or allergic reactions.The biosafety of the tested drug molecules after acute ocular exposure was tested on the eyes of Dutch belted rabbits (rotocols . Rabbit Glaucoma is one of the leading causes of irreversible blindness. Current drugs suffer from diminishment of activity and/or severe side effects upon prolonged usage. To address these problems, a new target, CACNA2D1, was identified, and PRG, which targets the protein, was found and showed a promising IOP-lowering effect in our previous studies. To further evaluate CACNA2D1 as a potential drug target, several other molecules binding to this protein were identified and tested for their in vivo activity.In our current study, to identify additional compounds that bind to CACNA2D1, a homology model was built and used to dock against the NCI library to find possible hits. Two compounds, namely, acivicin and zoledronic acid, stood out among the others and became the best candidates. This model was validated after testing these compounds in vivo for their IOP-lowering effect. Our results demonstrate that though they have lesser potency, these compounds share the same target protein and binding sites as PRG. Together, these data suggest CACNA2D1 is a validated drug target in treating glaucoma and worthy of further investigation for more potent compounds.


Addressing sustainable development goals to reduce neonatal mortality remains a global challenge, and it is a concern in Ethiopia. As a result, the goal of this study was to assess the incidence and determinants of neonatal mortality in the first 3 days among babies delivered in the referral hospitals of the Amhara National Regional State.A hospital-based prospective cohort study was conducted among 810 neonates in the first 3 days of delivery between March 1 and August 30, 2018. The neonates were followed up from the time of admission to 72 h. Interviewer-administered questionnaires and medical record reviews were conducted for data collection. Data were entered into Epi-data manager version 4.4 and analysed using STATA™ version 16.0. The neonate’s survival time was calculated using the Cox-Proportional hazards model.The overall incidence of neonatal mortality in this study was 151/1000 births. Neonatal mortality was significantly higher among neonates whose mothers came between 17 and 28 weeks of gestation for the first visit; among those whose mothers labour was not monitored with a partograph, mothers experienced postpartum haemorrhage and developed a fistula first 24 h, and experienced obstructed labour. However, 39% were less risky among neonates whose mothers were directly admitted and whose mothers had visited health facilities in less than 1-h, both.This study revealed that approximately 1 in 7 neonates died within the first 3 days of life. The determinants were the timing of the first antenatal visit, quality of labour monitoring, maternal complications, and delay in seeking care. Thus, scaling up evidence-based interventions and harmonising efforts to improve antenatal care quality, promote institutional deliveries, provide optimal essential and emergency obstetric care, and ensure immediate postnatal care may improve neonatal survival.The online version contains supplementary material available at 10.1186/s12884-021-04122-8. The newborn period is critical in the miracle of life and death. In 2018, the global neonatal mortality rate was estimated to be 18 deaths per 1000 live births, with 28 deaths per 1000 live births in Sub-Saharan Africa, and in 2016, it was 30 deaths per 1000 live births in Ethiopia . In EthiIn low and middle-income countries, it has been suggested that the scaling-up of evidence-based interventions that begin during the antenatal period, coordinated efforts aimed at improving the quality of antenatal care, promoting institutional deliveries, providing optimal essential and emergency obstetric care, and ensuring immediate postnatal care of neonates are essential , 6. ConsAlthough the perinatal mortality rate definition starts at 28 weeks of gestation in low and middle-income countries (LMICs), the mortality rate is higher than the developed countries that stated the definition from 20 weeks of gestation. The perinatal mortality rate includes all stillbirths and neonatal deaths in a given period over the total number of births multiplied by thousands . PreviouNeonatal mortality rates and determinants in the early neonatal period (the first 3 days of life in this case) are essential for arranging programs and identifying suitable interventions. Local and recent evidence about the spread of the problem could help implement programmatically relevant decision-making . HoweverThis study was conducted at the maternity wards of referral hospitals in the Amhara Regional State between March 1 and August 30, 2018. The National Regional State is located between 9° 20′ and 14° 20′ North latitude and 36° 20′ and 40° 20′ East longitude in the Northwestern part of Ethiopia. According to the Central Statistics Agency, the projected total population estimate Amhara Region in 2020/21 is 22,536,999 . Of these, 20.08% were urban residents  than those who started the initial antenatal care visit before 16 weeks of gestation. Mothers not monitored with a partograph during labour were 2.66 times the risk of neonatal mortality  compared to their counterparts. Direct admission was 39% less risk of neonatal death than  those admitted from referral to another health facility.Maternal complications within 24 h were also a significant risk factor for newborn mortality. Mothers experiencing postpartum haemorrhage were about three times risky for new-borns death , and those who developed fistula in the first 24 h were also about four times risky for new-borns death . Obstructed labour was more than twice risky  for neonatal mortality and less than 1-h maternal first delay in visiting health facility was 39% less risk of neonatal death   , South AIn our study, 122 neonates were lost within the first 3 days of life, giving an overall neonatal mortality rate of 151/1000 total deaths and a stillbirth rate of 103.7/1000 total births. This figure shows a significant decline from a study conducted in the Tikur Anbesa specialised hospital (225/1000 live births)  and (302Regarding the determinants of neonatal mortality within the first 72 h, gestational age at the first antenatal care visit was a risk factor. Women who came between 17 and 28 weeks of gestation for the first visit were 1.67 times more likely to lose their child than those who started the initial antenatal care visit before 16 weeks of pregnancy. This finding is consistent with studies conducted in Tigray regional state , Felege Maternal complications within 24 h were also a significant risk factor for neonatal mortality. Of these, the experience, postpartum haemorrhage, fistula development within the first 24 h, and obstructed labour were found to be three times, four times, and more than twice risky for neonatal death within the first 72 h of life. Our study’s findings regarding fistula and postpartum haemorrhage as risks for neonatal mortality were unique in this finding. The possible reason for neonatal mortality among mothers facing postpartum haemorrhage and fistula might be intrapartum asphyxia. In cases of maternal complications, the attention of health care providers diverts to saving the mother, and in some cases, neonates would not get adequate care, which leads them to intrapartum asphyxia. However, future research should be conducted to get the exact cause of neonatal mortality in such complications. However, this study’s results, which identified obstructed labour as a risk of neonatal mortality, were similar to studies conducted in Hawassa University hospital, Ethiopia , and terMoreover, mothers who were not monitored with partograph during labour were nearly three times the risk of neonatal mortality than their counterparts. This result was supported by a study in Addis Ababa  and TigrFurthermore, direct admission was 39% less risk of newborn mortality than those admitted from referral to another health facility. In other words, mothers who require a referral were either suffer from severe obstetric problems or transfer time. We extend the time to receive skilled care. Besides, less than 1 h of maternal first delay to visit health was 39% less risk of neonatal death. This result was similar to a study in Tigray Northern Ethiopia, showing that seeking skilled care at the start of labour was protective for perinatal mortality , Uganda Despite the indications of the Ghanian study , this stWe hypothesised high neonatal mortality in tertiary care centres in the first 3 days of life and found that about 1 in 7 newborns died in the first 3 days of life in the tertiary level of care in Northwestern Ethiopia. The leading causes of newborn death were neonatal jaundice, followed by complications like birth trauma, congenital anomalies, and asphyxia.Moreover, the determinants of neonatal mortality were delay for the first ANC visit, more than 1-h maternal first delay to visit a health facility, and health system-related determinants such as not monitoring labour with, admission by referral were the significant factors. Furthermore, obstetric determinants were mothers’ experience of postpartum haemorrhage, fistula development within the first 24 h, and obstructed labour. Therefore, each hospital in the region must start implementing the WHO recommendation on positive pregnancy experience for addressing the health system and obstetric risk factors of newborn mortality. Additionally, awareness creation and adherence to the recommended level of care are essential.Additional file 1.


Aspergillus niger for three weeks. This common soil fungus has shown exceptional ability to alter the distribution and mobility of selenium in the presence of both solid phases. The fungus initiated the extensive bioextraction of selenium from the surfaces of amorphous ferric oxyhydroxides, while the hausmannite (Mn3O4) was highly susceptible to biodeterioration in the presence of selenium. This resulted in specific outcomes regarding the selenium, iron, and manganese uptake by fungus and residual selenium concentrations in mineral phases as well. The adverse effects of bioleaching on fungal growth are also discussed.Bioleaching of mineral phases plays a crucial role in the mobility and availability of various elements, including selenium. Therefore, the leachability of selenium associated with the surfaces of ferric and manganese oxides and oxyhydroxides, the prevailing components of natural geochemical barriers, has been studied in the presence of filamentous fungus. Both geoactive phases were exposed to selenate and subsequently to growing fungus  Iron and manganese oxides and (oxy)hydroxides belong to geochemically active phases that are involved in the trapping of trace elements in soils and sediments . This isSelenium is a trace element that plays an important role in human health ,8. HowevAlthough the manganese and ferric oxides’ and (oxy)hydroxides’ surfaces represent excellent natural active sites for selenium immobilization ,16,17, iAspergillus niger, which is one of the most studied fungal strains regarding the issue of bioleaching of natural and synthetic mineral phases.Since selenium is not considered a global contaminant, there is a lack of scientific effort and focus on studying the fungal effects on selenium mobility in the presence of manganese and ferric oxides and (oxy)hydroxides in comparison to other potentially toxic oxyanions, including chromium, arsenic, and antimony. This inspired us to study the alterations in selenium distribution and mobility in the presence of the common soil fungus Aspergillus niger CBS 140837 strain. The fungus was isolated from mercury-contaminated soil .Samples were characterized by X-ray powder diffraction (XRD) analyses elsewhere ,23 and w−1 selenium (VI) concentration, prepared from Na2SeO4 p.a. . Therefore, the final pulp density of ferric oxyhydroxides and hausmannite was 2.2 g·L−1, with the total iron and manganese concentrations of 1.4 g·L−1 and 1.6 g·L−1, respectively. The final concentration of total selenium in the selenium(VI)-treated media was 50 mg·L−1. The suspensions were stirred at 130 rpm for 24 h; one portion was then sampled for the total selenium concentration in both solid phases and medium, and the other portion was subsequently inoculated with fungal spores, followed by the static three-week incubation at 25 °C in the dark.Series of 45 mL culture Sabouraud Dextrose Broth Media  in 100 mL Erlenmeyer flasks autoclaved at 121 °C for 15 min were supplemented with 0.11 g of amorphous ferric oxyhydroxides or hausmannite. Except for selenium-free control experiments, all treatments were supplemented with 5 mL of 500 mg·LThereafter, the fungal biomass was separated from the culture media, washed with distilled water, and dried at 25 °C. The spent culture media were then filtered through a filter membrane with 0.45 μm pore size. Dry biomass weight and pH values  of each culture filtrate including controls were determined. The residual non-dissolved solid phases were air-dried and digested. The biomass, non-dissolved residue (after digestion), and the spent culture media were analyzed for total selenium, manganese, and iron by inductively coupled plasma–optical emission spectrometry (ICP-OES) or flame atomic absorption spectrometry (F AAS). Control experiments were carried out without either selenium or ferric oxyhydroxides and hausmannite, following the same protocol. All treatments were in triplicate.Culture filtrate, dried biomass, and insoluble residue were analyzed for total selenium content by ICP-OES at line Se I 196.090 nm using an ICP spectrometer Jobin Yvon 70 Plus  equipped with a concentric nebulizer  and cyclonic spray chamber; plasma power: 1000 W. The accuracy of the method was checked using certified reference materials BCR-185 (bovine liver) and NCS DC73350 . The results were in agreement with certified values within their uncertainties. The precision of the method was 5% (relative combined standard uncertainty with coverage factor k = 2).−1 single-element standard solutions . The accuracy of the method was checked using certified reference materials NIST SRM 1577c (bovine liver), NCS DC73350 , and CRM Astasol AN9090MN . The results were in agreement with certified values within their uncertainties. The method’s precision was 4% for manganese and 6% for iron (relative combined standard uncertainty with coverage factor k = 2).The total manganese and iron content were analyzed by F AAS at lines Mn 279.5 nm and Fe 248.3 nm using an AAS spectrometer PerkinElmer Model 1100 . Deuterium background correction was used for both elements. Calibration solutions were prepared from CertiPUR ICP 1000 mg·LExcept for the amorphous ferric oxyhydroxides-treated media, the presence of selenate resulted in a statistically significant decrease of pH at the end of the three-week cultivation a. Since The negative effects of selenium on fungal growth were statistically significant in comparison to the control, regardless of the addition of solid inorganic phases b. Howeve−1 of manganese was recorded in the culture media in the absence of selenium, its presence triggered the manganese release from hausmannite whose dissolution rate was five times higher in comparison to selenium-free treatment. However, there was only a marginal difference in the concentration of iron bioextracted from the amorphous ferric oxyhydroxides in both selenium presence and absence with the average values of 0.19 g·L−1 and 0.16 g·L−1, respectively  at the initial day of inoculation to 0.4 mg  after three weeks of incubation b. SeleniA. niger has shown the ability to bioaccumulate selenium and iron, and manganese as well. While there was no statistically significant difference in comparison of selenium concentration in biomass of the control treatment and biomass incubated in the presence of ferric phases (on average of 1.1 mg·g−1), manganese phases statistically significantly increased the amount of bioaccumulated selenium (2.4 mg·g−1)  . On the  mg·g−1) .−1 sorption capacity, which is similar to other reported values, e.g., the maximum sorption capacities of 15.1 mg·g−1 and 22.5 mg·g−1 reported for nanoparticles of ferric oxyhydroxides and hydrous ferric oxides impregnated in a polymeric matrix, respectively [A. niger significantly decreased the ability of ferric phases to immobilize selenium since its average content in ferric amorphous oxyhydroxides decreased by 70% after cultivation  are produced in high quantities in media under iron limitation. Therefore, the contribution of siderophores to the biodeterioration of ferric phases is questionable, since the medium used in this study was not iron deficient, and these metabolites were not quantified. Similarly, the citrate also seems to be produced to overcome the iron limitation. However, the surplus of saccharose may facilitate citrate production via overflow metabolism [A. niger is an efficient acidifier of its surroundings via the exudation of protons and a well-known producer of other low-molecular-weight organic acids, e.g., oxalate, whose extrusion depends on the ambient pH [−1 , the growth inhibition of fungus was expected. The toxic effect should have been mitigated by the presence of solid ferric/ferrous iron and manganese oxides and oxyhydroxides [It was reported that the 0 mg·L−1 . Therefodroxides  that woudroxides . Thus, wdroxides , could rHausmannite’s effect on selenium mobility in the presence of filamentous fungus is contrasting the behavior of selenate in media treated with amorphous ferric oxyhydroxides. Its affinity toward selenium is significantly lower, and thus, the bioextraction is also less pronounced . BalistrBeauveria caledonica to heavy metals led to overexcretion of citric and oxalic acids, with the former reported to actively contribute to the reductive dissolution of manganese oxides in acidic solutions [Interestingly, the presence of selenium triggered the bioleaching of hausmannite. Thus, the concentration of free manganese in the medium increased more than five times compared to selenium-free treatment a. Since olutions .A. niger strain, which was reported elsewhere [−1 concentrations of dissolved manganese in the medium oxides’ bioleaching, especially regarding selenium mobility and bioaccumulation.In summary, the common soil filamentous fungus


Abnormal uterine bleeding requires the investigation of the endometrium. Histology is typically used but there remains room for the improvement and use of cytology.Women presenting for clinically indicated office endometrial biopsy were prospectively enrolled. Tao endometrial brushing and office endometrial biopsy were performed, and surgical procedure if clinically indicated. Tao brush cytology specimens were blindly reviewed by up to three pathologists, consensus obtained, and scored as: benign, atypical (favor benign), suspicious, positive for malignancy, or non‐diagnostic. Cytology and histology were compared to surgical pathology to determine sensitivity, specificity, positive, and negative predictive values to detect AH  or EC .Clinical indications of 197 enrolled patients included postmenopausal bleeding , abnormal uterine bleeding , and abnormal endometrium on ultrasound without bleeding . Of the 197 patients, 185 (93.9%) had cytology score consensus and a total of 196 (99.5%) had consensus regarding cytology positivity.Surgical pathology diagnoses (N = 85) were 13 (15.3%) FIGO grade 1 or 2 EC, 3 (3.5%) AH, and 69 (81.2%) benign endometrium. Sensitivity and specificity to detect EC or AH were 93.7% and 100%, respectively, via endometrial biopsy; 87.5% and 63.8%, respectively, via endometrial cytology when scores of malignancy, suspicious, or atypical were considered positive.In a high‐risk population, Tao brush endometrial cytology showed high sensitivity to detect AH and EC comparable to biopsy histology when considering scores of malignancy, suspicious, atypical, and non‐diagnostic. Revisiting the potential value of endometrial cytology in the contemporary era of endometrial diagnostic workup is warranted. Tao brush cytology is a sensitive tool for endometrial cancer evaluation in symptomatic women. There is a high concordance of Tao brush readings amongst blinded pathologists Diagnosis of endometrial pathology by this sampling method has traditionally required a pathologist that specializes in cytologyAt present, direct comparison performance data between endometrial cytology and endometrial biopsy are lacking. Although early data suggest that endometrial cytology as a diagnostic for AH and EC has acceptable performance,22.1Women presenting to the Abnormal Uterine Bleeding Clinic for a clinically indicated office endometrial biopsy were prospectively enrolled between February 2013 and August 2015, in the feasibility period of a large ongoing prospective cohort study.Specimen collection and processing:Endometrial tissue obtained via office endometrial biopsy was fixed in formalin. Endometrial biopsy pathology was interpreted per clinical standard of care by pathologists. After Tao endometrial brushing was completed, the bristled tip of the brush was removed from the Tao device using a wire cutter and placed in PreservCyt. In the cytopathology laboratory, the brush was scraped and the resulting material was placed back into the same PreservCyt vial. A monolayer cytology slide was made after glacial acetic acid treatment, using the Thin Prep 2000 Processor (Hologic) and Papanicolaou stained. Each sample was pre‐screened by a cytotechnologist, and then randomly assigned to be blindly reviewed by up to three of five study pathologists . Each sample was scored as: benign, atypical (favor benign), suspicious, positive for malignancy, or non‐diagnostic (did not meet criteria for another score) Figure . All stu2.2Data were summarized using standard descriptive statistics. Endometrial biopsy histology diagnoses of AH, EC, or non‐diagnostic were grouped as positive. Cytology diagnoses from Tao brush sampling were analyzed with two different categorizations: positive for malignancy, suspicious, or atypical were considered positive for criteria A; and positive for malignancy or suspicious were considered positive for criteria B. Diagnostic performances of cytology and office endometrial biopsy were compared to the criterion standard of surgical pathology to determine sensitivity, specificity, positive, and negative predictive values. Ninety‐five percent confidence intervals were constructed using exact methods for a binomial parameter. A two‐sided McNemar's test for comparing correlated proportions was used to compare sensitivity (and likewise specificity) between cytology and endometrial biopsy. Statistical analysis was performed using the SAS version 9.4 software package .33.1One hundred and ninety‐seven patients met the study inclusion criteria and had an adequate endometrial biopsy along with Tao Brush sampling. The mean age among the 197 patients was 55.0 (SD 7.6) years with 98 (49.7%) postmenopausal , but the statistical power was limited with only 16 patients with EC or AH on surgical pathology. The office endometrial biopsy was significantly more specific than Tao brush sampling for criteria A (p < 0.001) but not for criteria B (p = 0.11).Among the 85 patients with all 3 specimens, sensitivity and specificity of office endometrial biopsy to detect EC or AH were 93.7% and 100%, respectively. For the endometrial cytology from the Tao brush sampling, sensitivity and specificity were 87.5% and 63.8%, when scores of positive for malignancy, suspicious, or atypical were considered positive (criteria A in Table 4The high sensitivity (87.5%) of Tao brush cytology in this study supports further evaluation of endometrial sampling by brushing as a useful diagnostic approach for AH and EC in symptomatic women undergoing outpatient clinic evaluation. Our findings are consistent with previously reported dataThe Tao brush is a device that has been suggested to provide a more complete sampling of the endometrial cavity due to the flexibility and design of the device.The Tao brush cytology specimen is processed in a very similar manner to cervical cytology, which has been an established diagnostic method for several decades. Cervical cytology techniques continue to evolve with the incorporation of automation, artificial intelligence, and ancillary molecular tests (such as HPV) that have improved test performance.Meanwhile, the field continues to advance in the area of potential molecular testing  which may serve to complement endometrial cytology in the future.One of the strengths of this study was that each cytology specimen was reviewed by up to three pathologists with routine cytopathology experience but without extensive experience or training in endometrial cytology. This suggests that the learning curve for endometrial cytology may not be a limitation in the further development of endometrial cytology as a diagnostic test. The strength of interpretation was enhanced by the consensus of two pathologists; however, this is not routine in clinical practice for cervical cytology and may add expense due to additional pathologist time required if incorporated into clinical practice. To our knowledge, this is the first study assessing pathologist consensus among endometrial cytology specimens. Additionally, to our knowledge, this study is the largest thus far of women whose office endometrial biopsy and Tao endometrial brushing biospecimens have undergone direct comparison as well as the comparison to surgical specimen.This present study was subjected to the areas of limitation. As previously mentioned, obtaining consensus was a study advantage, but the use of multiple pathologists may limit the generalizability to centers with solo practice or insufficient pathologists for multiple reviews. It is possible that multiple reviews could be eliminated with increased pathologist experience or automated processes. In our study, at least 45.7%  of patients had a biopsy performed first. This may have disrupted the endometrium enough to allow greater tissue collection by the Tao brush and could cause a false elevation in sensitivity. Using criteria A, the sensitivity of Tao brush for EC or AH was 100% (7/7),75% (6/8), and 100% (1/1) for those with endometrial biopsy first, Tao brush cytology first, and unknown timing, respectively . However, these numbers are too small to draw any meaningful conclusions. Collection bias was also likely to present given that only patients who underwent clinically indicated surgery for diagnostic or therapeutic indications  were included in the cohort utilized to compare office collection techniques. Thus, final diagnoses demonstrated a higher proportion of EC and AH (nearly 16% combined) than would be expected in the setting of AUB and PMB. Caution should be utilized drawing conclusions in this area as this study was limited by the number of final EC and AH diagnoses.In summary, given the promising sensitivity of the Tao brush in our study and the consistency among studies comparing this sampling method to endometrial biopsy, further study on its role as a triage and diagnostic method for the work‐up of AUB and PMB is warranted. With its potential for superior comfort, ease to learn, and cost‐effectiveness, endometrial cytology may offer distinct advantages above endometrial biopsy for the patient, for the provider, and for the health system as a whole. Given contemporary advances in molecular assays and automated cytology processing and analyses, revisiting endometrial cytology as a diagnostic tool is warranted.Dr. Bakkum has received Mayo Clinic CCSG grant and V Foundation funding. Dr. Bakkum and Dr. Wentzensen both have funding from the Intramural Research Program of the National Cancer Institute (grant number Z01CP010124‐21). Additional authors have no conflict of interest to disclose.All authors contributed to conceptualization, data curation, project administration, investigation, writing review, and editing.


Neuronal ceroid lipofuscinosis type 2 (CLN2) disease is an autosomal recessive inherited neurodegenerative lysosomal storage disorder caused by deficient tripeptidyl peptidase 1 (TPP1) enzyme, leading to progressive deterioration of neurological functions commonly occurring in children aged 2–4 years and culminating in early death. Atypical cases associated with earlier or later symptom onset, or even protracted course, have already been reported. Such variable manifestations may constitute an additional challenge to early diagnosis and initiation of appropriate treatment. The present work aimed to analyse clinical data from a cohort of Latin American CLN2 patients with atypical phenotypes.Experts in inborn errors of metabolism from Latin America selected patients from their centres who were deemed by the clinicians to have atypical forms of CLN2, according to the current literature on this topic and their practical experience. Clinical and genetic data from the medical records were retrospectively revised. All cases were presented and analysed by these experts at an Advisory Board Meeting in São Paulo, Brazil, in October 2018.Seizures, language abnormalities and behavioural disorders were found as the first manifestations, appearing at the median age of 6 years, an older age than classically described for the late infantile form. Three novel mutations were also identified.Our findings reinforce the inclusion of CLN2 in the differential diagnosis of children presenting with seizures, behavioural disorders and language abnormalities. Early diagnosis will allow early initiation of specific therapy. Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative lysosomal storage disorder caused by deficient activity of the tripeptidyl peptidase 1 enzyme (TPP1).The classic form (or late infantile), in which the onset of symptoms frequently occurs nearly 2–4 years, is the most well‐documented phenotype. This form includes language difficulties, seizures, sleep disturbances, ataxia, movement disorders, motor deterioration, dementia and visual loss, culminating with precocious death.In this cohort, patients with atypical CLN2 disease presented with seizures, language abnormalities and behavioural disorders as first symptoms.The median age of symptoms onset was 6 years, an older age than classically described for the late infantile form of this disease.Three novel mutations were identified in these patients.TPP1 gene result in the classical late infantile phenotype, with symptom onset between 2 and 4 years of age, including seizure, progressive neurodegeneration, visual failure and motor impairment.Neuronal ceroid lipofuscinosis type 2 (CLN2) is an autosomal recessive neurodegenerative lysosomal storage disorder caused by deficient activity of the tripeptidyl peptidase 1 enzyme (TPP1). This disease is part of a group of progressive neurogenetic disorders, the neuronal ceroid lipofuscinoses (NCLs), also known as Batten disease.TPP1 gene. Either demonstration of deficient TPP1 enzyme activity in leukocytes or fibroblasts, or detection of two pathogenic mutations in trans are considered diagnostic for CLN2 disease.The presentation of the classic disease form is generally considered to be consistent and uniform, being well defined by age of onset (late infantile) and multiple neurological signs and symptoms, as well as specific electrophysiological findings and pathological changes in magnetic resonance images.There are no cures for the NCLs yet. Many therapeutic attempts have been proposed, including enzyme supplementation, gene therapy, stem cell therapy, anti‐inflammatories and small molecules – but most of these are currently in early stages of clinical development.Patients diagnosed with CLN2 disease may have a disease course that deviates from the typical clinical pattern. This atypical form is variable and broad in terms of age of onset, course of disease evolution, and multiple phenotypes, suggesting that there is a spectrum for this disease in terms of symptomatic presentation. Some of the atypical cases exhibit significant parkinsonian features, later age of onset, slower progression, and, remarkably, absence of seizures and visual loss.A recent survey of 25 CLN2 cases in South America showed that patients with a more severe phenotype  had null TPP1 activity, while the group of patients with residual TPP1 activity showed later onset and longer life‐span, with slower progression, less epilepsy syndrome and a higher frequency of ataxia particularly at a late stage of the disease.Although the existence of atypical forms of CLN2 disease is recognised by experts in the field, description of the natural course of this form of the disease, especially in Latin America, remains overlooked. Therefore, the present study was tailored to collect data from patients with atypical CLN2 disease in Latin America, attempting to identify potential patterns of clinical presentation, enzymatic activity and/or genetic findings that could assist physicians anywhere to reach a diagnostic suspicion of CLN2 more effectively.A group of 13 Latin American experts in the field of inborn errors of metabolism were invited to participate in an Advisory Board meeting supported by BioMarin in São Paulo, Brazil, in October 2018. Before the meeting, these experts provided medical data from patients with atypical forms of CLN2 in their centres using a spreadsheet with multiple clinical variables such as first symptoms, age at disease onset, enzyme activity, diagnostic methods and genetic profiling. Data were presented and analysed by these experts throughout the Advisory Board meeting.Inclusion criteria consisted of presenting with the atypical phenotype of CLN2. For that, an atypical phenotype is represented by earlier or later symptom onset, varied symptoms and/or slower progression but the maintenance of neurodegeneration and possible premature death.n), mean, standard deviation, median, minimum and maximum values.After the meeting, the spreadsheets were revised to collect additional data and statistical analysis was performed. Descriptive statistics was used to perform the analysis, using frequencies and percentages to represent categorical data variables. Continuous data variables were summarised by the number of subjects  of the patients with documented age of the first symptom onset experienced at least one additional symptom before their next anniversary (i.e. at the same age of the first symptom). The most frequent concurrent symptoms were cognitive decline  and language difficulties . Interestingly, the majority of the patients  who displayed more than one symptom within 12 months had seizures as the first symptom.n = 23, min = 1, max = 46) and these were: seizures , movement disorders  , ataxia , behavioural abnormalities , cognitive impairment , language regression  and visual impairment . Table The symptoms that motivated parents towards a medical consultation occurred at 7 years on average  of the patients in the cohort. Most abnormalities  were detected using low‐frequency photostimulation (1–3 Hz range).Different diagnostic methods were employed Table . All patGene panels for identification of genetic mutations were used in 47% of the patients 14/30); whole exome sequencing was used in 23% (7/30) and other methods  were used in 37% of the patients (11/30). Diagnostic confirmation was achieved by both identification of genetic mutation and TPP1 enzymatic activity in 23% (7/30) of the patients, and exclusively by enzymatic activity assay in 20% (6/30) of the patients, as shown in Table 0; whole The most frequent mutations in this cohort were c.887‐10A > G  and c.1048C > T . One patient presented as homozygous for the intronic mutation c.827A > T and three novel mutations were detected in our population: c.1226G > A, c.1343C > A and c.1552‐1G > A.In this cohort, 30% (9/30) patients have affected siblings  median age at symptom onset was 6 years; (ii) the most common first symptoms were seizures (47%), followed by language abnormalities (20%) and by behavioural abnormalities (17%); (iii) during the disease course, the most common symptoms were: language difficulty (100% of patients); cognitive impairment (93%), seizures and ataxia  and (iv) three novel mutations were found in the studied population.The classic phenotype of CLN2 is associated with seizures, movement disorders and ataxia.TPP1 gene . Interestingly, this patient had a very early disease onset with seizures at 1 year of age, progressing with visual loss, myoclonus and cognitive decline. Results of the metabolomic analysis of this patient will be published separately in a case report.Early diagnosis of classic CLN2 disease still constitutes a challenge despite the development of clinical diagnostic algorithms and the availability of CLN2 genetic testing and TPP1 enzyme assays.Complete correlation between specific mutations associated with CLN2 disease and the various phenotypes is not yet available and challenged by the large number of mutations described, the epidemiology of the disease (rare condition) and the variability in clinical presentation.The c.1048C > T (cln2.139) mutation was described in six patients of this cohort . There is evidence that this is a disease‐associated variant, since it has been reported in homozygous form in an Argentinian patientThree novel mutations were detected in our population: c.1226G > A, c.1343C > A and c.1552‐1G > A; the impact of these mutations on TPP1 structure, activity and phenotype remains to be determined.The limitations of the present study include limited sample size, the retrospective nature of data as well as limiting data retrieval in clinical charts. In this regard, for example, we could retrieve general information as ‘language abnormalities’ from the chart instead of specific details of the clinical manifestation.Atypical presentation of rare diseases such as CLN2 poses additional challenges to early diagnosis. Data from this cohort of Latin American patients with atypical CLN2 show that potential differences between atypical and classical phenotypes include a later onset of disease, and that motor manifestations may not be so common as the first symptoms. In light of the availability of specific therapy that may alter disease progression, these findings strengthen the importance of clinical suspicion and investigation of CLN2 even in patients above the classical age of the disease onset who present mainly with seizures but also language or behaviour abnormalities.


Plasmodium falciparum (Pf) sporozoite (SPZ) malaria vaccines. A large, pivotal trial of the safety and efficacy of the radiation-attenuated Sanaria® PfSPZ Vaccine against P. falciparum is planned for 2022. This study assessed the incidence of malaria at the phase 3 study site and characterized the influence of socio-demographic factors on the burden of malaria to guide trial design.Extensive malaria control measures have been implemented on Bioko Island, Equatorial Guinea over the past 16 years, reducing parasite prevalence and malaria-related morbidity and mortality, but without achieving elimination. Malaria vaccines offer hope for reducing the burden to zero. Three phase 1/2 studies have been conducted successfully on Bioko Island to evaluate the safety and efficacy of whole A cohort of 240 randomly selected individuals aged 6 months to 45 years from selected areas of North Bioko Province, Bioko Island, was followed for 24 weeks after clearance of parasitaemia. Assessment of clinical presentation consistent with malaria and thick blood smears were performed every 2 weeks. Incidence of first and multiple malaria infections per person-time of follow-up was estimated, compared between age groups, and examined for associated socio-demographic risk factors.There were 58 malaria infection episodes observed during the follow up period, including 47 first and 11 repeat infections. The incidence of malaria was 0.25 [95% CI ] and of first malaria was 0.23 [95% CI ] per person per 24 weeks . Incidence of first malaria with symptoms was 0.13 [95% CI ] per person per 24 weeks . Multivariate assessment showed that study area, gender, malaria positivity at screening, and household socioeconomic status independently predicted the observed incidence of malaria.P. falciparum malaria.Despite intensive malaria control efforts on Bioko Island, local transmission remains and is spread evenly throughout age groups. These incidence rates indicate moderate malaria transmission which may be sufficient to support future larger trials of PfSPZ Vaccine. The long-term goal is to conduct mass vaccination programmes to halt transmission and eliminate  Plasmodium falciparum (Pf). Children and pregnant women are the most vulnerable for dying of the disease, and progress with burden reduction has plateaued [The burden of malaria is concentrated in sub-Saharan Africa, where 94% of the estimated 229 million malaria cases occurred in 2019, the large majority caused by lateaued .P. falciparum sporozoite (PfSPZ) malaria vaccine approach, with radiation-attenuated Sanaria® PfSPZ Vaccine and the chemo-attenuated Sanaria® PfSPZ-CVac, the two leading products under development [The Government of Equatorial Guinea (EG) is partnering with Medical Care Development International (MCDI), Ifakara Health Institute (IHI), Swiss Tropical and Public Health Institute (Swiss TPH), and Sanaria Inc. to evaluate the safety and efficacy of the whole elopment , 5. PfSPThree phase 1/2 studies of PfSPZ Vaccines have been conducted in EG. The first study was the first clinical trial in the history of EG and evaluated the safety and tolerability of PfSPZ Vaccine in adults . The secAn efficacious pre-erythrocytic vaccine, like PfSPZ Vaccine, which interrupts malaria transmission since the development of asexual blood stages and associated sexual stages is prevented . TherefoInformation on the expected incidence of malaria and malaria with symptoms on Bioko Island is needed to properly design the upcoming trial, including estimating the appropriate sample size , 10. HenThe study was conducted in communities in an urban/peri-urban area of Malabo District in the Bioko North Province on Bioko Island, EG, where the capital city, Malabo, is located. The population of Malabo District is estimated to be 191,671 individuals living in 68,306 households with an average household size of 2.8 individuals . The population is composed of several ethnic groups, including the Bubi, Fang, Annobones, Ndowe, Bisio, and Fernandino. The study area  . The maievalence –15.Anopheles melas and Anopheles coluzzii [The major vectors maintaining residual transmission on Bioko are coluzzii . Significoluzzii . Since 2coluzzii . Despitecoluzzii , 18.For this study, communities with high malaria prevalence (parasitaemia above 15% by rapid diagnostic test (RDT)) based on a 3-year average (2016–2018) of data from the annual cross-sectional malaria indicator survey were selected. The selected communities were mainly from the western and southwestern regions of the Malabo District; they were categorized based on the administrative boundaries and geolocation into four study areas (area I–IV)  augmented by assessing any acute febrile illnesses (passive surveillance). Information on co-morbidities that occurred was collected from participants during study visits through updates on medical history, physical examination, and laboratory investigations. The study targeted the enrollment of 240 participants split evenly into three age groups: 6–59 months, 5–17 years, and 18–45 years.Permission was obtained from local government officials and community representatives to use the household database from the BIMCP/NMCP to identify household members to be invited to participate in the study . SubsequA multistage sampling was done, first communities were selected primarily due to relatively high parasite prevalence in non-travelling children aged 2–14 years and proximity to the Sampaka Health Centre (no more than 6 kms from the facility) based on the BIMCP/NMCP 2018 health census. Individual demographic information from the 15 selected communities, was aggregated to the household level to determine the number of eligible individuals per age group permanently residing in inhabited households within the study area. Households with at least one eligible individual were included in the sampling frame, representing 20,240 eligible individuals residing in 7901 eligible households.Thereafter, Python 2.7, PANDAS package was used to randomly select and order individuals from unique households for each of the three study age groups. The number of individuals to sample within each of the communities was pre-determined to ensure a representative geographic distribution of sampled individuals proportionate to community sizes. Once an age group was filled, individuals were only invited to participate if contributing to the unfilled age groups.Potential adult participants, or the parents or guardians of potential pediatric participants, present during household visits were asked to come to SHC to provide written consent, 9–17-year-olds were asked to provide written assent, and 6–8-year-olds were asked to provide verbal assent. Screening evaluation included the history of previous illnesses, and physical examination included vital signs and anthropometric measurements. Blood samples were collected for analysis of malaria parasitaemia , the latter performed retrospectively), complete blood count with differential, and biochemistry including alanine aminotransferase (ALT), aspartate aminotransferase (AST), total bilirubin, creatinine, and glucose. A urine sample was collected for human chorionic gonadotropin pregnancy testing in women aged 9–45 years. Socio-demographic data were also collected.Participants were considered eligible for the study if they were permanent residents of the study area, age between 6 months and 45 years, and provided consent/assent. Participants were excluded if they had a history or clinical manifestations of serious or chronic disease requiring frequent medical care including human immunodeficiency virus (HIV) infection, clinical tuberculosis, sickle cell disease, malignancies, diabetes mellitus, hypertension, mental illness, or seizures. Additional exclusion criteria were mid-upper arm circumference (MUAC) < 11.5 cm for children under 5 years of age , grade 3 abnormal laboratory parameters , pregnancy, and history of allergy or serious adverse reaction to artemether/lumefantrine (AL). Participants with issues that increased the risk of non-adherence to study procedures, including the intention to move from the study area during the study period, were excluded.® Cipla Ltd, India) according to manufacturer’s instructions [Eligible participants were given a directly observed curative 3-day, six-dose course of artemether–lumefantrine (AL)  tubes and kept in a cooler box to maintain ambient indoor temperature until reaching the laboratory.Participants were treated based on TBS results. For those TBS positive participants who were at the clinic, treatment with an anti-malarial was initiated before they were allowed to go home. For those who were seen at home they were invited back to the clinic by the community health workers and were then reviewed again at the clinic and an anti-malarial treatment initiated.Compliance with study visits and procedures was encouraged by the provision of a study calendar to participants, by using multiple contact methods , by home visits by community mobilizers, and by provision of transportation to the study center. Participants were compensated for time spent during scheduled study related clinic visits (approximately 12 United States dollars), but not for home visits.Study doctors were available to see study participants 24 h–7 days a week. Participants were encouraged to visit the SHC or call the study doctors whenever they were sick. Medical history, physical examination and laboratory investigations were done as needed for clinical evaluation. All participants with history of fever had their blood checked for malaria by TBS, RDT and later qPCR.Plasmodium genes namely the Pan-Plasmodium 18S RNA sequence (Pspp18S) and the P. falciparum-specific acidic terminal sequence of the var genes (PfvarATS). The human Ribonuclease P gene (HsRNaseP) was used as a DNA extraction and qPCR amplification control. All qPCR assays were run in duplicate and both non-template control (Molecular grade nuclease-free water) and P. falciparum 3D7 DNA were included as negative and positive controls, respectively. The sample was considered positive if the quantitation cycles (Cq) for each of the two replicates was Cq < 40 for Plasmodium spp. gene and Cq < 28 for qPCR amplification control. Haematology parameters were established using ABX Pentra 60 C+  and biochemistry parameters using Cobas Integra 400 Plus . Urine pregnancy tests were done using Hexagon hCG 1-step rapid tests .Thick and thin blood smears and the RDTs for assessment of malaria parasites were prepared from samples collected during home visits or at the study clinic. These were prepared and independently analysed at the SHC laboratory to allow blinding and standardization of the preparation and reading of the results. All remaining study related laboratory analyses were done at the public health laboratory in Baney which is located 24 km from the SHC. TBSs were prepared and read as described previously . In shorR. Data were entered and verified by independent teams.Information was collected on paper-based study case reports forms and these were entered into a customized electronic database, Castor EDCThe design assumed an incidence of new malaria infections (parasitaemia) over 24 weeks of 10% for each age group. In order to detect an incidence rate of 10% per person per 24 weeks with a relative precision of 20% (from 8 to 12%) for each age group at 90% confidence level, and considering a 15% rate of loss to follow up, a sample of 80 participants was targeted for recruitment in each age category.Data were analysed using STATA version 15 . Descriptive statistics were used to summarize the data. Household socioeconomic status (SES) was calculated as a weighted sum of data on household possessions and utilities, using principal components analysis and the scores divided into quintiles. Elements included in the measure: ownership of radio, TV, bicycle, refrigerator, mobile phone, computer, bed net, and source of energy . The incidence rate of first infections was computed for 24 weeks of follow up as the total number of first infections observed after enrolment divided by total person time at risk.P. falciparum infection divided by the total number of participants at the start of follow up for each age category.Person-time was censored for loss to follow up or identification of a first infection. Incidence rate for multiple infections was computed for the 24 weeks of follow-up as the total number of malaria infections developing after enrolment divided by the total person-time at risk. Twenty-eight days were censored off from time at risk after each treatment of malaria infection. Kaplan Meier survival curves were used to compare time to first infection between groups, and Cox regression analysis was used to explore factors associated with first incidence of malaria infection. Lastly, proportional incidence of infection was computed as the number of participants developing first Following the ordered sampling frame of participants identified from the BIMCP/NMCP household database who lived in the selected study areas see “”, 545 weThe median age in years of those enrolled was 9.80 with range (IQR) from 1.02 to 42.51  10.0, 18.8), 6 participants in group 1, 11 participants in group 2, and 16 participants in group 3  per person per 24 weeks follow-up. The overall incidence of first infection was 0.23  per person per 24 weeks. The observed incidence rate for first infections was similar in the three age categories: 0.22 , 0.26  and 0.20  infections per 24 weeks in groups 1, 2 and 3, respectively (Table Incidence of infection appeared to be greater in: (1) males, 0.31 95% CI 0.22, 0.45) versus females, 0.16 , the trend observed in all areas, and (2) those living in Areas III, 0.26  and IV, 0.39  compared to those in Areas I, 0.14  and II, 0.12 . Incidence was lowest in the highest socioeconomic group , 42% (n = 10) and 47% (n = 8) of the total cases of malaria recorded in groups 1, 2 and 3 respectively. Of these, 27 were first cases of malaria with symptoms. The incidence rate for all cases of malaria with symptoms was 0.14  and for first malaria with symptoms was 0.13 . The incidence rates of first cases of malaria with symptoms in groups 1, 2, 3 were 0.16 , 0.10 , 0.11 , respectively. Younger children and those living in Area IV tended to have more clinical episodes, but the numbers were small and the differences between areas were not statistically significant  and 157.48 , respectively. The geometric mean parasite density for all cases of malaria with symptoms was 1898 (95% CI 431–8364), 357 (95% CI 88–1449) and 112 (95% CI 26–493) for age groups 6 to 59 months, 5 to 17 years and 18 to 45 years, respectively.Common illnesses that occurred in the study participants included respiratory tract infections, gastroenteritis, diarrhoea, and skin infections. About 70%  of illnesses required medical attention during the 24 weeks of follow up, and the young age group had more events compared to the older age groups  had significantly higher burden compared to the more urban locations. This heterogeneity indicates that malaria control interventions have variably impacted malaria prevalence and morbidity depending on the setting; importantly, however, nowhere have they interrupted transmission, a situation shared with many other settings in Africa –24. The Furthermore, significant local transmission remains despite the impact of the study itself, which, due to active surveillance and prompt treatment, could be expected to reduce the incidence of malaria infection in the participants beyond the effects of ongoing control measures, as has been observed in several sites in Africa , 26. HenThe relatively similar incidences of both asymptomatic and symptomatic malaria among the age groups suggests that intensive control has shifted the age pattern of the risk of infection away from children compared to before the intervention , as seenSurprisingly, the results of this study indicate there was a significantly higher risk of malaria infection in males than in females, but no higher risk for other illness events including pneumonia and diarrhea. This sex difference in malaria infection, contrasting with similar studies in moderate transmission areas in Africa , extendeTravel has been documented to be an important contributing factor to the maintenance of local transmission in Malabo, and Bioko Island as a whole , 35. HowAs expected, the incidence of malaria with symptoms was lower (13%) compared to asymptomatic infections (20%), likely due, at least in part, to intense follow up and prompt treatment  as the mOverall, there were few illnesses during follow up which is similar to what has been observed in other studies in Africa where good clinical care is provided as part of a study , 26. As This study was conducted to serve as a baseline for the conduct of a large safety and efficacy study of PfSPZ Vaccine that has so far shown promising results in EG and elsewhere , 7, 40. The study provided a good platform for capacity development, building the skills and confidence of the research team; the ability to conduct assessments of malaria incidence will be critical for tracking the success of future efforts to eliminate malaria from Bioko Island.Intensive malaria control efforts in Bioko Island have resulted in a large reduction in the prevalence of malaria; local transmission remains, however, and the risk of infection is now spread relatively evenly through all age groups, a finding which is consistent with areas that have changed from high to lower transmission areas. The remaining burden of malaria offers an opportunity to properly evaluate the efficacy and safety of PfSPZ Vaccine and establish the safety and efficacy data required to receive marketing authorization (licensure).