Most experiments studying bacterial microbiomes rely on the PCR amplification of all or part of the gene for the 16S rRNA subunit, which serves as a biomarker for identifying and quantifying the various taxa present in a microbiome sample. Several computational methods exist for analyzing 16S amplicon sequencing. However, the most-used bioinformatics tools cannot produce high quality genus-level or species-level taxonomic calls and may underestimate the potential accuracy of these calls. We used 16S sequencing data from mock bacterial communities to evaluate the sensitivity and specificity of several bioinformatics pipelines and genomic reference libraries used for microbiome analyses, concentrating on measuring the accuracy of species-level taxonomic assignments of 16S amplicon reads. We evaluated the tools DADA2, QIIME 2, Mothur, PathoScope 2, and Kraken 2 in conjunction with reference libraries from Greengenes, SILVA, Kraken 2, and RefSeq. Profiling tools were compared using publicly available mock community data from several sources, comprising 136 samples with varied species richness and evenness, several different amplified regions within the 16S rRNA gene, and both DNA spike-ins and cDNA from collections of plated cells. PathoScope 2 and Kraken 2, both tools designed for whole-genome metagenomics, outperformed DADA2, QIIME 2 using the DADA2 plugin, and Mothur, which are theoretically specialized for 16S analyses. Evaluations of reference libraries identified the SILVA and RefSeq/Kraken 2 Standard libraries as superior in accuracy compared to Greengenes. These findings support PathoScope and Kraken 2 as fully capable, competitive options for genus- and species-level 16S amplicon sequencing data analysis, whole genome sequencing, and metagenomics data tools. Characterizing the composition of microbial samples commonly relies on the amplification of 16S ribosomal subunit sequences, a ubiquitous gene with highly conserved regions. The subunit simplifies efforts to isolate and amplify 16S rRNA with established PCR primers and hypervariable regions to establish identity and phylogeny. 16S rRNA and rDNA sequencing can be used to identify known prokaryotic species and act as a proxy to quantify the relative abundances of operational taxonomic units (OTUs) within microbiome samples.High-throughput sequencing has greatly accelerated the study of microbiomics, that is, the scientific field focused on studying the composition, diversity, and function of microbial communities and their interactions with their hosts or environments2, current profiling software for 16S amplicon sequencing data hesitates to identify down to the species level. Instead, they cluster reads based on sequence similarity to assign genus or higher-level identifications to increase specificity and sensitivity, or they directly use error-filtered sequences for taxonomic classification4. As the capabilities of modern sequencing platforms increase, and as bacterial reference genome databases expand and improve, more potential arises for achieving enhanced 16S analysis performance with alternative methods more commonly applied in whole genome metagenomics.Methods for taxonomic profiling of ribosomal RNA gene sequences enable sample OTU identification by classifying rRNA sequences into taxonomic groups. While considerable accuracy in species-level identification is attainable with available tools4, QIIME 25, its predecessor, QIIME 26, and Mothur7. QIIME 2 and Mothur were both originally developed shortly after the invention of next-generation sequencing and, along with QIIME 2, essentially follow the same workflow: reads are typically clustered de novo based on sequence similarity into operational taxonomic units (OTUs) or denoised OTUs (many refer to these as amplicon sequence variants or ASVs) depending on whether complete sequence identity is desired for clustering. The initial clustering step serves to 1) improve computational efficiency by limiting the number of sequences needing alignment to a large set of reference genomes and 2) accommodate the low levels of genetic variation present within a given bacterial strain, thereby mitigating sequencing errors. For nearly a decade, the cutoff for OTU inclusion was 97% sequence identity9, but current cutoff recommendations are now around 99–100% sequence identity10, typically after some form of denoising or other correction for sequencing errors11.The most common software packages currently employed in analysis of 16S amplicon sequencing data are DADA212. PathoScope employs a Bayesian mixed modeling framework to reassign ambiguously aligned reads, dampening potential sequencing errors and minor genetic variation14. As another alternative, Kraken 2 performs alignment-free k-mer searches against a reference genome library15 and makes taxonomic assignments to each read based on the cumulative number of k-mer matches across an entire read against each taxonomic node in its reference library. By bypassing a sequence clustering step, PathoScope and Kraken 2 individually avoid the potential pitfalls inherent in OTU generation and denoising errors17, although remain susceptible to sequencing errors. While DADA2, QIIME 2, Mothur, Greengenes, and SILVA are all tools designed to address the specific needs of 16S amplicon sequencing, improvements in sequencing technologies, expanding bacterial reference genome databases, and increased availability and affordability of computational resources have collectively made many of the specific issues addressed by these tools irrelevant. Meanwhile, the increased flexibility and power of a tool such as PathoScope may yield augmented results despite being computationally intensive and designed to fulfill a more general metagenomics purpose19.An alternative to OTU clustering includes directly aligning reads against a reference genome library, as is done by PathoScope 2.023. The most commonly used reference databases for 16S amplicon analyses are Greengenes24, SILVA25, and the Ribosomal Database Project (RDP)26. Each database exclusively contains 16S rRNA gene sequences and offers taxonomic information for each reference sequence. SILVA is well maintained and releases updates regularly, although as of this writing, the most recent update is SILVA 138.1 . Meanwhile, Greengenes has been stagnant for years; its most recent update at the time of submission was Greengenes 13_8, released in August 2013. As a result, Greengenes lacks several essential bacteria, including Dolosigranulum species27, implicated as playing a protective role in preventing disease in human airways29. It is worth noting that a new version, Greengenes230, was made available in 2022 amidst the review process for this paper, and was subsequently not included in this paper’s analyses. Although QIIME 2 and Mothur are compatible with any reference genome library, QIIME 2 uses Greengenes by default, and Mothur’s documentation  recommends SILVA. DADA2 maintains reference databases for SILVA, RDP, and Greengenes, with the flexibility to create custom databases. Kraken 2 has its own curated “Standard” bacterial library, with a taxonomic tree based on NCBI’s taxonomy database by default31, and has also released Kraken 2-compatible formatted versions of Greengenes, SILVA, and RDP. The current PathoScope reference library recommendation is to download the complete RefSeq representative genome database32, a collection of curated high-quality bacterial genomes and assemblies. RefSeq is constantly updated, and as such, results of any analysis using RefSeq as a reference library may vary according to the date of the library download.These profiling methods all rely heavily on the quality of the reference library used, as has been shown in previous benchmarking studiesGiven these considerations, we systematically benchmarked several current community profiling tools and reference libraries created for both metagenomic and 16S analysis. We evaluated the tools QIIME 2, Mothur, PathoScope 2, and Kraken 2 in conjunction with reference libraries from Greengenes, SILVA, Kraken 2, and RefSeq. Using several publicly available 16S sequencing datasets of synthetic mock communities, we specifically analyzed genus- and species-level performance across pairs of profilers and libraries. We tested 136 samples comprising varying species richness and evenness, several different amplified regions within the 16S rRNA gene, and both DNA spike-ins and cDNA from collections of plated cells. Our evaluative comparisons utilized a combination of diversity and accuracy-based measures to determine what methods and tools provided the best performance in profiling 16S amplicon sequencing datasets.33; 33 samples are from Kozich et al.34; 29 samples are from Fouhy et al.35; and 5 samples are from Karstens36. These datasets are hereafter referred to as the Lluch, Kozich, Fouhy, and Karstens samples. The species compositions for each set are delineated in Table 136 mock community sequencing samples were collected in total from four publicly available sequencing datasets and analyzed in our evaluation. 69 samples are from Lluch et al.We evaluated five analysis pipelines applied to the 136 mock community samples: DADA2, QIIME 2, Mothur, PathoScope, and Kraken 2.For the standalone implementation of DADA2, all samples were filtered and trimmed, with errors learned on forward and reverse reads. Learned errors were used to conduct inference on predicted error presence across all reads as a denoising measure. Paired reads were merged and chimeras were removed, with taxonomy assigned down to the species level. When filtering and trimming, for most samples we used the parameters maxN = 0, maxEE = c, truncQ = 2, rm.phix = TRUE, and tlength = 0. For unpaired reads, we set maxEE = 3. Several Kozich samples suffered from extreme quality degradation at read ends; to correct for this, we set tlength = c. Finally, when running the Fouhy Ion Torrent samples, we set the DADA2 function parameters HOMOPOLYMER_GAP_PENALTY = − 1 and BAND_SIZE = 32. For the filter and trim step, we also set trimLeft = 15. These settings were based on recommendations for processing Ion Torrent data in the DADA2 FAQ.For all QIIME 2 analyses, we used the DADA2 plugin to cluster sequences and construct feature tables. We decided to test the DADA2 plugin alongside its standalone package due to the broad user base of QIIME 2. However, the standalone implementation uses 100% sequence identity over Mothur and QIIME 2 (with 97%) and uses exact sequence matching rather than a k-mer based method (as in the QIIME 2 q2-feature-classifier). All mock datasets could be run with paired-end reads besides the Fouhy datasets. In most cases, DADA2 did not require truncation of paired-end sequences and only the initial 6 bp were trimmed from each read. However, quality scores at the end of nine samples from the Kozich dataset were universally low enough  to require truncation to 240 bp for forward reads and 200 bp for reverse reads for Kozich samples. Taxonomy was assigned using custom naïve Bayes classifiers constructed for each set of mock community samples based on their amplified 16S region. The QIIME 2 artifact output files were converted into BIOM format and subsequently into tab-delimited text format for downstream analyses and pipeline comparisons.For Mothur analyses, all recommended procedures were followed according to Mothur documentation where possible. For paired-end sequences, the native make.contigs function was used to join reads. In the pre.cluster step of Mothur analysis, the “diffs” parameter  was set to 2 for joined sequencing reads shorter than 250 bp, 3 for joined reads of length 250–349 bp, and 4 for longer joined reads. For cluster.split, we set the “taxlevel” parameter to 4, with a “cutoff” of 0.03.rentrez.For PathoScope 2.0 analyses, Bowtie2 alignment parameters were set to “–local -R 2 -N 0 -L 25 -i S,1,0.75 -k 10 –score-min L,100,1.28.” These values were optimized for 16S sequencing reads, requiring higher similarity to a reference genome to be considered a hit than the default settings due to the highly conserved nature of portions of the 16S rRNA gene. Phylogeny for each taxon was inferred from the NCBI taxon id (ti) for each reference genome using the entrez_fetch function from the R package For Kraken 2 analyses, Kraken 2 taxonomic reports were created for each sample. These were parsed into a taxon/feature counts matrix that included the complete phylogeny for each identified taxon as reported by Kraken 2.We used five bacterial sequence reference databases in conjunction with the aforementioned pipelines: Greengenes 13_8, SILVA 138, two versions of RefSeq’s representative genomes, and the Kraken 2 Standard library . According to the Kraken 2 manual, the Kraken 2 Standard library is compiled using the RefSeq database, so it could be considered analogous to the RefSeq2020 library. The RefSeq libraries were downloaded on November 2, 2018, and June 23, 2020; these are denoted as “RefSeq2018” and “RefSeq2020.” Greengenes and SILVA are specifically 16S reference databases as they include only sequences for the bacterial 16S rRNA gene. RefSeq2018, RefSeq2020, and the Kraken 2 Standard database are all whole-genome libraries, with no special modifications for use with 16S amplicon sequencing data.We analyzed 136 mock community samples using a total of 11 distinct pairings of analysis tools and reference libraries: DADA2 only with SILVA, QIIME 2 with Greengenes and SILVA, Mothur only with SILVA (the default reference library), PathoScope using Greengenes, SILVA, RefSeq2018, and RefSeq2020, and Kraken 2 with its Standard library, SILVA, and Greengenes. While the SILVA database includes species-level taxonomic information for most of its representative 16S sequences, note that Mothur collapses feature counts into genus-level clades and thus does not make species-level calls. The adaptations of SILVA used for Kraken 2 and QIIME 2 did not provide species-level calls. Thus, only eight of the 11 pairings make species-level calls. The pairings and pipeline parameters are summarized in Table Bacillales order would be given the metadata: “phylum: Firmicutes, class: Bacilli, order: Bacillales, family: o_Bacillales, genus: o_Bacillales, species: o_Bacillales.”A counts matrix was created from the results of each of the 11 pipeline/reference pairs for each operational taxonomic unit (OTU), denoised OTU, and feature. Each feature was assigned phylum, class, order, family, genus, species, and subspecies level information when available. For a given database, whenever a taxonomic label was missing, the lowest level taxonomy available for a feature was propagated using that database’s taxonomic path, taking note of what granularity was available (taxonomic best hit). For example, a feature assigned only as a member of the Several metrics were used in assessing the overall quality and power of each 16S analysis pipeline and reference library at each taxonomic level. Results were independently evaluated at each taxonomic level. Any reads or features not assigned to a taxon at a given phylogenetic level were excluded from analysis, except where otherwise specified.The metric of taxon detection sensitivity is defined here as the portion of expected taxa in a mock community sample detected by a given pipeline, at a minimum of 0.1% relative abundance. It essentially examines how often a given method can correctly determine an organism’s presence in the mock community.Read assignment specificity is defined here as the portion of reads from a given sample assigned to taxa that are actually present in that sample’s mock community. This is equivalent to 1 minus the portion of reads assigned to spurious taxa. This metric identifies the frequency of read assignment to incorrect organisms for a given method.wi and ti are respectively the measured and true read counts of taxon i. We evaluated the union of the expected and detected taxa for each sample, using The Normalized Root Mean Squared Error (NRMSE) was calculated as the root mean square error normalized with the assumption that the variance could be increasing given higher read counts. For each sample’s results, given by the equation5 index. The R package vegan37 was used to calculate the Shannon and Simpson indices. The R package breakaway was used to calculate the breakaway_nof138 index, which predicts both the number of unobserved taxa and the number of true singletons based on the non-singleton frequency counts. Due to the alpha diversity metrics' sensitivity to library and count size differences39, we converted the relative abundances of the mock community samples' ground truths to virtual sequencing libraries of 1,000,000 reads again using the vegan37 package. A rarefaction depth of 10,000 reads per sample was used to normalize all samples and ground truth libraries.To assess each pipeline’s ability to estimate the true alpha diversity within a sample regardless of accurate species identification, we calculated the log-fold change between the expected and the measured alpha diversity as measured by the Shannon index, the Simpson index, and the breakaway_nof1lme440, and post hoc comparisons were performed with the lsmeans function from the R package lsmeans41. These LMMs examine the relevant performance metric as the measured variable, using the 136 mock community samples as a random effect and the pipeline/reference library pair as a fixed effect.A series of linear mixed-effects models (LMMs), coupled with post hoc least-square means tests and a Tukey multiple comparison correction, were used to determine which pipelines outperformed each other in sensitivity, specificity, error rates, and alpha diversity estimates. LMMs were estimated using the lmer function from the R package Figure At the genus level Fig. A, DADA2 Bifidobacterium adolescentis, Prosthecobacter fusiformis, and Clostridium beijerinckii.Generally, taxon detection sensitivity was lower at the species level than at the genus level Fig. B. MethodAt the genus level, the average read assignment specificity was generally lower for Kraken 2 with its Standard library ; PathoScope and QIIME 2 with Greengenes ; and DADA2, Kraken 2, and Mothur with SILVA . , which were significantly lower than all methods . These were significantly lower than all other error rates  and Simpson  alpha diversity indices, with significantly higher deviations than all other methods . PathoScope generally matched the true Shannon indices more closely than all other methods . On the other hand, Kraken 2 using its Standard library and SILVA frequently overestimated the number of species present by several orders of magnitude , performing worse than all other methods  and that PathoScope (regardless of reference library) and Kraken 2 (with its Standard library) dominate the upper right quadrant, where sensitivity and specificity are both high. In particular, PathoScope excels in both sensitivity and specificity when used with either SILVA or RefSeq2018. Similarly, Fig. Overall, no single pipeline or reference library performed the best in all evaluative metrics, but some holistic trends are present, especially at the species level. Figure 42, causing some species to be underrepresented or even absent in the collected cDNA libraries from a plate43. While 16S amplification primers are designed to bind to universal conserved regions of the 16S rRNA gene, there is still clearly some amplification bias during PCR44. Contamination from reagents45, local bacteria in the air, on gloves, or in a pipette tip box can further complicate matters. Thus, limitations of different experimental conditions and methods can dramatically affect the quality of results obtained from mock communities. Present sequencing errors and contamination suggest that amplicon reads will not identify with taxa as precisely as might a tidy, evenly distributed set of sequences drawn from a closed set of well-characterized species. It should then be evident that no analysis pipeline could theoretically exist to perfectly measure a mock community. Such a feat would require identifying only the expected species in their exact proportions, with no extraneous observations. As such, the proximate best method to analyze 16S amplicon sequencing data is one that identifies the makeup of the microbiome as truthfully as possible. Mock microbial communities can provide a level testing ground for existing tools to find their relative strengths and weaknesses in performance.Mock bacterial communities, either derived from spike-in DNA sequences or extracted from mixtures of bacterial cell monocultures, provide a semblance of a “ground truth” to assess 16S amplicon sequencing analysis methods. Ideally, knowing which species at what amounts should be present in any genuine microbiome sample would allow for accurate identification in every analysis. There are, of course, complications in sequencing experiments: technical bias and errors are introduced into samples at every step of the experiment until being safely sealed as bits in a FASTQ file on a server. Mock species' relative abundances may be affected by subtle variations in pipetting technique as spike-in DNA is aliquoted from individual sources. Spike-in DNA might be cloned from mutated DNA, or an early PCR error may have propagated through an entire commercial stock of nucleic acids. Different species of bacteria vary in lysing difficulty46. This is likely due to several factors, including the small size of the Greengenes database in comparison to SILVA , and the fact that this version of Greengenes had not been updated since 201347.Of the pipelines tested, both QIIME 2 and Mothur were designed and built specifically for 16S amplicon sequencing analysis. Each has a suite of utility functions built to assist researchers in processing their data from the sequencer to differential abundance analysis and visualizations. Both are typically bundled with a dedicated bacterial 16S rRNA gene sequence database's reference library for alignment . However, our results present strong evidence that PathoScope and Kraken 2 outperform QIIME 2, Mothur, and DADA2, even when comparing reads to identical reference databases. This phenomenon interestingly occurs despite Kraken 2 and PathoScope's status as more general whole genome sequencing and metagenomics data tools. In pairwise comparisons, PathoScope is more sensitive and specific at taxon detection and has a lower error score than either DADA2, QIIME 2, or Mothur, and has comparable alpha diversity index estimates at both the genus and species levels. In general, SILVA’s outperformance of Greengenes confirmed results found in previous benchmarks of 16S amplicon sequencing analysis methodsKraken 2, when used with its Standard library, was rarely the top-performing analytic method in terms of sensitivity or specificity, although it was generally less error-prone than QIIME 2, Mothur, or any tool using Greengenes as a reference library. Kraken 2 has the added practical utility of being extremely fast and easy to use. Yet one constraint in analyzing Kraken 2 results is that they cannot be upsampled from a given taxon level, whereas PathoScope, QIIME 2, and Mothur all allow the tracing back of the taxonomic hierarchy of a given microbe. Both QIIME 2 and Mothur take advantage of naïve Bayes classifiers, which work most efficiently when trained on the specific region of the 16S rRNA gene amplified by PCR primers. Overall, PathoScope was the most sensitive in detecting taxa and specific in assigning reads, and the least error-prone tool when paired with either SILVA or RefSeq2018. However, it was not without limitations, as its computational expenses seemed to be an additional order of magnitude above those of other methods. This was evident from large interim SAM files (> 128 GB) and runtimes on the order of several hours, whereas Kraken 2 in particular took mere minutes. Issues aside, PathoScope is likely to outperform QIIME 2, DADA2, and Mothur in identification regardless of the database used. This finding partly results from PathoScope’s Bayesian mixed modeling identification algorithm, which accounts for the possibility that multiple species can be present in the sample or that the target strain is not present in the reference database. PathoScope consistently outperformed Kraken 2 in most cases, although the difference was often slight and not statistically significantly better. Overall, these comparisons show that methods designed for general metagenomics analyses consistently outperform methods specifically designed for analyzing 16S data.48. Compared to NGS, long-read sequencing technologies have been shown to perform better in classifying at the genus and species level50. We also observed differences in our study between the results of 15 samples sequenced with Ion Torrent sequencing, as compared to samples from the same mock communities that were sequenced using Illumina Miseq. Furthermore, a major limitation to 16S amplicon studies is that some clades of bacteria exist with identical 16S DNA in the commonly sequenced V4 region. These clades of difficult-to-identify bacteria make up the bulk of Kraken 2’s and PathoScope’s incorrect calls. For example, Bifidobacterium adolescentis was nearly universally misclassified by all methods as other Bifidobacterium species, and Prosthecobacter fusiformis was frequently misidentified as Prosthecobacter dejongeii, a species with which it shares over 99% of its 16S DNA sequence51. Even further complications arise from many bacteria having several copies of the 16S rRNA gene, which may not be identical between operons within a genome52. This latter point may be in part why metagenomic methods such as Kraken 2 and PathoScope outperform specific methods such as QIIME 2 and Mothur, especially at the species level. The metagenomic methods are better designed to account for multiple 16S rRNA genes if present.While many species are identifiable from their 16S rRNA gene sequence or a single hypervariable region, it is important to note that imperfect accuracy at this level is not solely a computational issue. For example, although the 16S rRNA gene is approximately 1550 bp long, the short sequencing reads obtained in most next-generation sequencing (NGS) only span about 250–500 bases and lack ideal resolution at the species levelEscherichia coli, PathoScope with RefSeq2020 reported the presence of Tumebacillus flagellates at relative abundances tightly correlated with the expected values of E. coli (Pearson’s r = 0.959). The circumstances strongly imply that reads actually originating from E. coli were incorrectly assigned to T. flagellates. T. flagellates is not even in the same phylum as E. coli, so the casual misassignment of reads between the species would be extremely unlikely based on 16S sequence similarity. Instead, a pairwise BLAST comparing E. coli’s 16S rRNA gene sequence to the T. flagellates scaffolds using the exact RefSeq entry that PathoScope had assigned those reads to (accession: NZ_JMIR01000093)53 revealed that one T. flagellates scaffold had a 100% identity alignment over 911 bp. The finding possibly represents a case of horizontal gene transfer of the 16S rRNA gene, but it appears far more likely that E. coli contamination existed in the DNA library, which then was sequenced and assembled into T. flagellates scaffolds. On further study, it became apparent that this is merely one example of pervasive sequence contamination, meaning the accidental inclusion of sequences from other organisms or the misclassification of sequences, in public genome databases. This phenomenon has been recently explored in the NCBI RefSeq database56. The recent prevalence of high throughput and the accelerating low cost of next-generation sequencing (NGS) technologies has led to a rapid increase in published genomes available in the RefSeq libraries, although imperfect methods and protocols for sequencing data are contributing to high contamination rates. Human contamination in published genomes, while not a problem in 16S analyses, is a particularly frustrating problem when analyzing shotgun metagenomics data. Clearly, metagenomic read-mapping approaches such as Kraken 2 and PathoScope afford the potential for the development of novel quality control pipelines for RefSeq and other genome sequence databases.One of PathoScope’s largest sources of error and lost taxon detection sensitivity calls when using the RefSeq2020 library comes from an apparent erroneous reference genome scaffold in the RefSeq representative genomes. In all mock community samples containing 60, but this is an ongoing problem that demands additional filtering and correction efforts after directly retrieving libraries from the public repository. Given the higher specificity and sensitivity of PathoScope when using the 2018 RefSeq library over the 2020 library, we recommend using older RefSeq libraries until newer versions have been processed to remove contamination. Also of interest is the high accuracy of SILVA in its species calls when using PathoScope, even though it cannot be used to make such calls when used with QIIME 2, Mothur, or Kraken 2. SILVA also presents a viable alternative to the RefSeq libraries in avoiding contamination.The increasing prevalence of poor sequencing quality control helps to explain why the RefSeq 2018 libraries often performed better than the 2020 libraries. Many tools have been developed to identify and correct contamination errors in sequences and public databases18. Miossec et al. found that among the tested pipelines, PathoScope 2.0 and Kraken represented high sensitivity and specificity in the benchmark results. However, we emphasize that further comparison of other metagenomics analysis pipelines such as MetaMix61, Centrifuge62, and Metaxa263 should be conducted to analyze their differential performance, especially as new methods are developed and published.We note that in performing this benchmark, we sought to evaluate several common 16S amplicon sequencing analysis pipelines alongside metagenomics analysis pipelines. 16S pipelines were chosen based on performance as well as prevalence in previously published benchmarks. To identify metagenomics pipelines, we used a previously published metagenomics benchmarking paper30 reference library as an phylogeny-based improvement over Greengenes. We also recommend PathoScope and Kraken 2 as fully capable, competitive options for conducting genus- and species-level 16S amplicon sequencing data analysis, in addition to outperforming other tools when using shotgun metagenomics dataDADA2, QIIME 2 and Mothur struggle to maintain accuracy at the genus level or granular species level in taxonomic analyses. Kraken 2, despite its primary purpose for whole genome sequencing metagenomics analyses, offers more power in analyzing 16S data without any increase in computational costs. PathoScope, while computationally more expensive, produces the most sensitive and accurate results of all evaluated pipelines when used on a diverse set of mock bacterial community samples. Analysis pipelines using SILVA as a reference library outperformed those using Greengenes significantly, and PathoScope using SILVA yielded the highest accuracies and sensitivities. While whole-genome reference libraries, such as Kraken 2’s Standard or RefSeq’s representative genomes, may provide some benefits over SILVA in terms of sensitivity, they may yield more spurious species-level calls. Based on the research conducted here with mock microbial communities, we recommend SILVA and RefSeq above other databases and strongly discourage usage of the Greengenes reference library for future analysis. While not included in our analysis due to release date, we do encourage users to try the Greengenes218.Supplementary Tables.


Surgical aortic valve replacement (SAVR) in female patients has been associated with higher mortality (up to 3.3–8.9%) and postoperative complication rates when compared with their male counterparts. In recent years, TAVI has been shown to provide a greater benefit than SAVR in women. We sought to assess the early outcomes of the contemporary aortic valve intervention practice  in patients referred to our cardiac surgery unit. The data of consecutive patients who underwent isolated aortic valve intervention for aortic valve stenosis during the 2018–2022 period were retrieved from our internal database. Several preoperative, intraoperative, and postoperative variables were analyzed, including the predicted risk of a prosthesis–patient mismatch. Nine hundred and fifty-five consecutive patients—514 women and 441 men—were included. Among them, 480 patients—276 female and 204 male—received a transcatheter procedure, and 475—238 women and 237 men—had conventional SAVR. The women were older and had higher EuroSCORE II, while the male patients presented a higher incidence of cardiovascular comorbidities. There was no difference in mortality or major postoperative complication rates after either the surgical or transcatheter procedures between the female and male populations. The availability and targeted use of different techniques and technologies have enabled the safe and effective treatment of female patients treated for severe symptomatic aortic valve stenosis with similar results when compared with their male counterparts. Aortic valve (AV) stenosis is the most common valve disorder due to an aging population . AlthougTranscatheter aortic valve implantation (TAVI) has provided an option for patients at medium/high risk ,7,8 or iThe combination of the peculiar clinical and pathophysiological features, anatomy characteristics, advanced age, and frailty typical of women presenting with symptomatic aortic valve stenosis poses several well-recognized therapeutic and technical challenges to both the surgical and TAVI approaches. At our cardiac surgery unit, we provide both surgical and transcatheter aortic valve interventions. At the beginning of our experience, TAVI was reserved for high-risk scenarios , but nowThis study reports our experience in aortic valve intervention—embedding both surgical and TAVI procedures performed by the same team of cardiac surgeons—and discusses the impact of this contemporary practice on early outcomes in female patients treated for severe symptomatic aortic valve stenosis.This is a single-center, retrospective analysis of prospectively collected data. All patients’ characteristics, intraoperative data, and periprocedural data were collected from the internal database of the Cardiac Surgery Unit at Lancisi Cardiovascular Centre—Polytechnic University of Marche–Ancona . The study was approved by the local ethics committee (CERM 2019 361), and informed consent was obtained in all cases.All consecutive patients who underwent a first-time isolated aortic valve intervention with biological prostheses (SAVR and TAVI) for aortic valve stenosis during the 2018–2022 period were included.Preoperative characteristics were defined according to EuroSCORE definitions . Outcome2/m2, moderate for values between 0.65 cm2/m2 and 0.85 cm2/m2, and severe if iEOA < 0.65 cm2/m2.The values for the effective orifice area (EOA) for each type of prosthesis implanted during the study period were retrieved from companies’ publications ,17, obseIn the surgical aortic valve replacement group, a minimally invasive approach including upper ministernotomy or right mini thoracotomy associated with an enhanced recovery protocol was our preferred strategy.Ministernotomy was the most used access. It was performed through a J-shaped incision (4–5 cm) from the sternomanubrial joint to the 3rd or 4th intercostal space. Cardiopulmonary bypass (CBP) was established through conventional central cannulation, and the right pulmonary vein was cannulated for left ventricle venting.Right mini thoracotomy (MT) surgery was performed through anterior right thoracotomy or trans-axillary access. The first was performed through a 4–5-cm skin incision at the 2nd and 3rd intercostal spaces, followed by costochondral cartilage dislocation. Trans-axillary access has been extensively described in previous publications and includes a 4–5 cm incision at the level of the 3rd intercostal space on the anterior axillary line . In thesThe key features of our enhanced recovery after surgery approach, besides reduced access and chest trauma, include the routine use of normothermia during standard and minimally invasive extracorporeal circulation (MiECC), combined with ultra-fast-track anesthesia with on-table extubation, proactive pain management, physiotherapy starting on day 0 postoperative, and immediate  patient–family contact ,25.TAVI procedures included either a transfemoral or a transapical approach.At the beginning of our program , the pro® and Sapien 3 Ultra ) and the Medtronic self-expandable ) prostheses. The prosthesis size was chosen according to the recommendation charts supplied by the companies, based on preoperative CT scan measurements.We used two platforms: the Edwards balloon expandable , while categorical variables are presented as numbers and percentages. Student p < 0.05.Statistical significance was defined as The analysis was generated using Statistical Analysis Software (SAS), Version 3.8, SAS University Edition .During the study period, 955 consecutive patients—514 women and 441 men—underwent an isolated aortic valve intervention for aortic valve stenosis with a biological prosthesis. Four hundred and eighty patients—276 female and 204 male—received a transcatheter procedure, and 475—238 women and 237 men—had conventional SAVR. p < 0.001; There was a progressive increase in the use of transcatheter procedures throughout the study period in both the female and male populations with TAVI performed in more than 75% of the patients during the last year (p = 0.009), with a concomitant reduction in EuroSCORE II values (p = 0.0015). Male SAVR patients exhibited a reduction in mean age throughout the study period, from 71 years to 68 years (p = 0.33), with no significant changes in EuroSCORE II (p = 0.73).During the study period, there was a significant decline in the mean age of female patients undergoing SAVR, from 74 years to 70 years , but no changes were appreciated in patients’ mean age . EuroSCORE II values reduced significantly during the study period for both female and male TAVI patients , a history of CAD, and previous cerebrovascular accidents was higher in the TAVI population than in the SAVR population.In the TAVI group, transapical and transfemoral approaches were performed, respectively, in 40 (14%) and 236 (86%) female patients and in 44 (22%) and 160 (78%) male patients. A balloon expandable prosthesis was used in 61% of cases in women and in 69% of cases in men. More than 75% of the female patients received a transcatheter prosthesis of ,25,26 siMinimally invasive access was used in more than 80% of the surgical cases for both the populations of female and male patients. Three surgical prostheses were used, as follows: the Carpentier–Edwards Magna Ease aortic valve  in 53% of women and 89% of men, the Intuity Valve System  in 25% of women and 2% of men, and the Trifecta bioprosthesis  in 22% of women and 9% of men. About 75% of the female patients received a surgical prosthesis size of ,22,23, wp = 0.81), 1.4% and 1.5%, respectively, after TAVI (p = 0.99) and 0.4% in both groups after SAVR (p = 0.99). Neurological events were reported, respectively, in four female patients (0.8%) and five male patients (1.1%) (p = 0.57). After TAVI, we registered a 1.1% rate of postoperative stroke in women and a 2.5% rate in men (p = 0.29). One female patient (0.4%) suffered a neurologic event after SAVR vs. no event in the surgical male population (p = 0.99).The overall in-hospital mortality rate was 1% in the female population and 0.9% in the male population . Women had a lower incidence of postoperative permanent pacemaker implantation and experienced a shorter stay in the ICU , nine after TAVI (4.4%) and three after SAVR (1.3%). As the population ages, the burden of aortic valve stenosis is increasing. This pathology seems to be occurring with a similar prevalence in male and female patients, but several data suggest that severe aortic stenosis in women is undertreated. Surgical series from national registries have included many more cases of SAVR in men than in women (63% vs. 37%) . Women rTAVI has emerged as an advantageous option for the treatment of aortic stenosis in women, for whom it provides a greater benefit than SAVR when compared with male patients ,28. NeveOur data did not confirm these findings. In the female SAVR population, we registered an in-hospital mortality rate and a postoperative stroke rate approaching 0% (0.4%), the need for CVVHD in 1.3% of cases, a median mechanical ventilation time of 5 h, and a median ICU stay of 24 h, with no difference when comparing these results with those of our male population. Similar satisfactory and comparable outcomes were found in female TAVI patients—hospital mortality at 1.4% and postoperative stroke at 1.1%—with a low occurrence of major vascular complications and severe paravalvular leak. The whole population of women who came to our attention for severe aortic stenosis—elderly (mean age: 80 years), symptomatic, and comorbid patients—received a safe treatment characterized by an in-hospital mortality rate of 1% and a stroke rate of 0.8%.The main reason for these results lies in the careful selection among a multidisciplinary Heart Valve Team of the most appropriate option, surgical or transcatheter, considering the peculiar advantages and limitations of these techniques and the available technologies and devices.At the beginning of our experience, the patients referred for a transcatheter procedure were older, frail, and at high risk for surgery . The enrAs a result, in our recent practice, TAVI has progressively outperformed conventional SAVR, addressing a larger number of elderly patients. In this way, we have been able to provide interventional treatment to more female patients who fully benefited from the greater advantages that transcatheter procedures appear to confer on women over AVR ,9,28 whiAlongside the presentation at an increased age and with higher numbers of comorbidities, a smaller body size with concomitant smaller cardiac structures, smaller aortic annuli, and the implantation of 19- and 21-mm-size surgical prostheses ,33 and ≤The prevention of PPM is important since it has been associated with worse hemodynamic function, less regression of left ventricular hypertrophy, and lower survival after SAVR ,36. AlthExpertise and proficiency in both surgical and transcatheter approaches can provide a comprehensive understanding of the potential anatomical and technical challenges typical of female patients with aortic valve stenosis. Tailored use of these techniques—with the availability of suprannular stented valves for which we favor a continuous suture technique and sutureless prostheses, balloon-expandable or self-expandable valves ,47, miniOur study shares the usual limitations associated with observational retrospective studies, although all the data were retrieved from our internal database, which is prospectively completed by specialist doctors before the discharge of patients. A peculiar limitation of our analysis lies in the ongoing debate about the correct assessment of PPM following TAVI or SAVR using measured or predicted iEOA. We have used predicted iEOA ,18,19,20This study was not designed to provide any comparison of outcomes between the two populations of patients who had surgical or transcatheter aortic valve intervention. Instead, we aimed to highlight the importance of mastering both techniques and providing the most appropriate treatment, based on patients’ characteristics and anatomy, in order to increase the offering of therapeutic options and deliver the best possible results to the whole referral population.No adjustment of preoperative characteristics was performed for the two populations of female and male patients since they differ a priori in disease presentation, natural history, and pathophysiology. Our study aims to provide an updated look at the early results in the treatment of women with aortic valve stenosis by comparing their outcomes with those observed in their male counterparts, fully considering their peculiar characteristics, which have historically been associated with poorer outcomes.We acknowledge that lowering the threshold of TAVI with a broad implementation of transcatheter techniques in younger and lower-risk patients with a longer life expectancy warrants future research focusing on late survival, functional status, and valve durability, as recent studies showing satisfactory results on valve durability at 6–8 years are still based on longitudinal data from octogenarian populations with limited survival during follow-up ,54.Women presenting with severe symptomatic aortic valve stenosis can nowadays be treated without excess operative risk and can receive aortic valve intervention as safe and effective as those expected in male patients. The availability of different techniques and technologies has successfully addressed the clinical, anatomical, and technical difficulties usually encountered in female patients and translated into improved hospital mortality and postoperative complication rates with enhanced hemodynamics after both surgical and transcatheter procedures with similar early results when compared with male populations.


We report a case of idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN) syndrome in a patient whose cystoid macular edema (CME) was successfully treated with aflibercept and pan-retinal photocoagulation (PRP). A 56-year-old male was sent to our uveitis service for further evaluation after a fluorescein angiogram revealed symmetric retinal ischemia for 360 degrees in both eyes. A fundus examination revealed an aneurysm, neuroretinitis, and occlusive vasculitis, all consistent with a diagnosis of IRVAN syndrome. An optical coherence tomography examination revealed CME of the left eye. A chest X-ray revealed minimally prominent interstitial markings. The patient had a positive QuantiFERON-TB Gold test and was treated for tuberculosis with a one-year course of isoniazid and pyrimethamine. A further workup for other infectious and autoimmune etiologies was negative. The initial treatment consisted of bilateral PRP of the areas of peripheral ischemia, treatment for which was provided in a fragmented fashion over the course of seven months. Soon after the diagnosis, he received treatment with two intravitreal injections of aflibercept (2 mg/0.5 mL), one month apart, to the left eye. Subsequently, four months following the presentation, he developed CME in the right eye, which was treated with a single intravitreal injection of aflibercept (2 mg/0.5 mL). At his last follow-up visit, four years after the initial presentation, the patient remained asymptomatic with 20/20 visual acuity in both eyes and no evidence of CME recurrence. Our case suggests that aflibercept may serve as an adjuvant to the standard treatment with PRP, especially in cases that present with associated macular edema. Idiopathic retinal vasculitis, aneurysms, and neuroretinitis (IRVAN) syndrome is a rare clinical entity of unknown etiology ,2. The A 56-year-old male was sent to our uveitis service for further evaluation of his peripheral retinal ischemia and disc anomalies. His past medical history was remarkable for an appendectomy, microcytic anemia, and abnormal glucose tolerance. His family history was remarkable for diabetes mellitus and hypertension. The review of systems and past social history were unremarkable.Upon ophthalmic exam, the patient’s best-corrected visual acuity was 20/20 in both eyes (OU) with a manifest refraction of +2.50 −0.75 × 100 in the right eye (OD) and +3.00 −1.00 × 085 in the left eye (OS). Intraocular pressure was 12 mmHg OU. A gonioscopy revealed open angles with no evidence of neovascularization OU. A slit-lamp examination was remarkable for nasal progressive pterygium of the conjunctiva OS and a 2+ nuclear sclerotic cataract OU. The anterior chamber was deep and clear, and there was no evidence of vitreous cells. An examination of the fundus revealed multiple aneurysms, lipid exudate, and occlusive vasculitis  OS. Two weeks subsequent to the intravitreal injection of aflibercept, a PRP was performed in a fragmented fashion on the peripheral areas of nonperfusion OU. The following month, the patient received a second intravitreal injection of aflibercept (2 mg/0.5 mL) OS and showed a notable reduction of edema. After six weeks, he began receiving monthly PRP laser sessions  that alternated between both eyes.After being in continuous follow-up for seven months, the patient developed cystoid macular edema (CME) superonasal to the fovea OD, which was treated with a single intravitreal injection of aflibercept (2 mg/0.5 mL) and resulted in significant improvement of his edema. A FA revealed stable ischemia and the resolution of the disc and macular leakage. Four years after the initial presentation, the patient’s symptoms resolved, his vision remains 20/20 OU, and there is no clinical evidence of CME recurrence.The etiology of IRVAN remains idiopathic. However, inflammation appears to be integral to its pathophysiology. Retinal vasculitis, aneurysmal dilations, and neuroretinitis are the three primary criteria of IRVAN, while peripheral capillary nonperfusion, retinal neovascularization, and macular exudation comprise the minor criteria . A liteThe inflammatory reactions that occur in IRVAN lead to focal ischemia of the blood vessel wall, thus causing increased vessel permeability and intimal collagen remodeling. This reaction makes IRVAN syndrome susceptible to aneurysmal artery dilatation. The aneurysmal dilatation is fragile and may be prone to rupture, which would therefore provoke hemorrhage and possible exudation of the vessel’s contents throughout one or more layers of the retina, leading to visual loss . The efPeripheral ischemia due to capillary nonperfusion can also promote the production of VEGF, which increases vascular permeability, resulting in macular edema and induced neovascularization . An abnAflibercept has some advantages that have been hypothesized to contribute to its increased efficacy since, compared to other anti-VEFG medications, it is an intermediate-sized molecule ,14. ItsAdditionally, the standard treatment of PRP was administered to curtail the metabolic demand of the areas of ischemia in the retina since various articles advised the use of PRP to prevent neovascularization and resolve retinal aneurysms and leaks ,17-19. These findings are in concordance with those of the study of Mansour et al., in which management with intravitreal injections of aflibercept plus heavy PRP was associated with a lower rate of treatment failure compared to management with a single treatment . SimilarOur case suggests that aflibercept may serve as an adjuvant to the standard treatment with PRP, especially in cases in which macular edema is present. Despite our patient’s successful response to the described treatment, prospective studies are needed to assess its effectiveness and safety.


Physical activity and sleep monitoring in daily life provide vital information to track health status and physical fitness. The aim of this study was to establish concurrent validity for the new Opal Actigraphy solution in relation to the widely used ActiGraph GT9X for measuring physical activity from accelerometry epic counts (sedentary to vigorous levels) and sleep periods in daily life. Twenty participants (age 56 + 22 years) wore two wearable devices on each wrist for 7 days and nights, recording 3-D accelerations at 30 Hz. Bland–Altman plots and intraclass correlation coefficients (ICCs) assessed validity (agreement) and test–retest reliability between ActiGraph and Opal Actigraphy sleep durations and activity levels, as well as between the two different versions of the ActiGraph. ICCs showed excellent reliability for physical activity measures and moderate-to-excellent reliability for sleep measures between Opal versus Actigraph GT9X and between GT3X versus GT9X. Bland–Altman plots and mean absolute percentage error (MAPE) also show a comparable performance (within 10%) between Opal and ActiGraph and between the two ActiGraph monitors across activity and sleep measures. In conclusion, physical activity and sleep measures using Opal Actigraphy demonstrate performance comparable to that of ActiGraph, supporting concurrent validation. Opal Actigraphy can be used to quantify activity and monitor sleep patterns in research and clinical studies. Limited physical activity and poor sleep are related to several neurological diseases and result in reduced quality of life ,2,3,4. RWearable technology with tri-axial accelerometers has been successfully used to measure physical activity and sleep for decades, and provides a cost-effective and practical solution for continuous tracking of daily life health status ,19,20. CAlgorithms for computing activity counts in the earlier models of these devices have been published ,24,25,26Several studies demonstrated the validity of accelerometer-based physical activity and sleep measures by comparing them to in-clinic gold-standard assessment ,28,29,30Wrist-worn accelerometers have been validated for sleep detection as well. Cole et al. developed and validated automatic scoring methods to distinguish sleep from wakefulness based on wrist activity during overnight polysomnography. They reported that their algorithms correctly distinguished sleep from wakefulness 88% of the time . Neishab® V2 Solutions  have been used extensively in clinical and academic research to characterize gait, balance, and other aspects of mobility. Opal® V2 wearable sensors contain lightweight inertial measurement units, and can be deployed both in the clinic and remote settings to capture a broad range of digital movement outcomes. Recently, Clario has developed an actigraphy solution to quantify physical activity and sleep measures during daily life. This solution contains one Opal sensor, configured to use the triaxial accelerometer, in a low power mode sampled at 30 hz to collect actigraphy data continuously. The Mobility Lab® software contains algorithms that generate the objective measures of daily activity levels and sleep. In this pilot study, we aimed to validate the Opal Actigraphy measures of physical activity and sleep and assess their agreement with those obtained from two different models of ActiGraph. We chose ActiGraph as a reference because ActiGraph devices  have been among the most widely used and validated actigraphy devices for over two decades [Opal decades . The maiTwenty healthy subjects without neurological, musculoskeletal, or sleep disorders participated in the study. The experimental protocol was approved by the Institutional Review Board of the Oregon Health & Science University (eIRB # 15578). All the participants provided informed written consent.Participants wore 4 wearable devices; 2 on each wrist. One Opal and one ActiGraph GT9X were strapped together and placed on the non-dominant arm, and one ActiGraph GT3X and a second ActiGraph GT9X were strapped together and placed on the dominant arm. Participants were instructed to wear the devices continuously for 7 days (day and night) during their daily activities and sleep. Data collection was performed by team at OHSU, independent of the data analysis team at Clario.The sensor data were processed by Mobility Lab  and ActiLife (GT3X and GT9X) to obtain accelerometer epoch counts data and sleep periods  . The couConcurrent validity is established when a new test or instrument is compared against a previously validated test, often a “gold standard” or that most widely used in the field. In this study, we aim to support validation of Opal Actigraphy by comparing it to the current most widely used wrist-worn sensor in the industry, the ActiGraph. To make the comparison between the devices, we chose several analytic approaches to assess levels of reliability between the Opal and ActiGraph GT9X algorithms. Evidence of high reliability will suggest the concurrent validity of the Opal instrument. Furthermore, we predict that the level of reliability and agreement between the Opal and ActiGraph GT9X will be comparable to the level of agreement between the two ActiGraph devices (GT3X and GT9X), thus providing additional support to validation of the Opal-based actigraphy solution. To establish reliability and agreement, we used intraclass correlation coefficients (ICCs)  and Bland–Altman plots. Additionally, we calculated mean absolute percentage error (MAPE) to test the performance between Opal versus ActiGraphy GT9X, and ActiGraph GT3X versus Actigraph GT9X. The aim was to test if the difference between the MAPE of Opal vs. GT9X is comparable  to tThe datasets generated during and/or analyzed during the current study are available from the corresponding author on reasonable request.Twenty healthy subjects participated in the study. The mean age of the group was 56 years (standard deviation (SD) = 22 years), with an average height of 65.5 inches (SD = 5.8 inches) and an average weight of 72.6 Kg (SD = 16 Kg). Compliance was high for weekly recordings, with an average of 6.5 days  of recording.Freedson98 Algorithm: The ICC values show high reliability and similar range in both comparisons for all activity measures estimated by Freedson98. The ICC ranges across activity levels for Opal versus ActiGraph GT9X were 0.9953–0.9996, and for GT3X versus GT9Xwere 0.9899–0.9999 see . Figure FreedsonVM3 Algorithm: The ICC values show high reliability and a similar range in both comparisons for all activity measures estimated by FreedsonVM3. The ICC ranges across activity levels for Opal versus ActiGraph GT9X were 0.9916–0.9981, and for GT3X versus GT9X were 0.9957–0.9996 see . Figure Opal Actigraphy and ActiLife solution (ActiGraph GT3X and GT9X) generated activity counts, activity intensity levels, and sleep measures during daily life. Comparisons between Opal and ActiGraph GT9X and between ActiGraph GT3X and GT39 were used to assess levels of reliability and agreement. Collectively, in this pilot study, our results indicate high reliability and similar agreement between the Opal and ActiGraph models, and between the two ActiGraph models, lending support for establishing concurrent validity.Activity measures: Total activity counts were slightly underestimated by Opal, and overestimated by GT3X compared to GT9X. This is consistent with the findings by John et al. who observed an overestimation of counts by GT3X compared to GT9X on a treadmill protocol [protocol . In contprotocol .Sleep measures: GT3X has been shown to be a reliable and valid to measure sleep compared to a gold standard in-lab, i.e., polysomnography (PSG) [hy (PSG) ,30,40. Shy (PSG) ,30,40. Ihy (PSG) ,30,40. Ihy (PSG) ) and OpaWe observed that activity measures yielded higher reliability metrics from ICCs than sleep measures. Further, we observed that for some of the activity and sleep measures, Opal is underestimating while GT3X is overestimating compared to GT9X, and vice versa, with a notable outlier from the Bland–Altman plots. Both of these observed differences could be attributed to the different hardware, sensor characteristics see , and algThere are several limitations of the current study. First, we only assessed the reliability and agreement of activity and sleep measures of the Opal compared to ActiGraph in healthy middle-aged adults, and findings may not be applicable to other cohorts. Second, we only validated Opal’s activity and sleep measures with a small sample of subjects, therefore limiting the strength of the conclusions that can be drawn regarding agreement, especially from the Bland–Altman plots. Finally, we need to investigate the activity and sleep measures for the minimal clinically important difference in order to establish whether the difference between the Opal and GT9X is clinically meaningful. Hence, future studies are needed to strengthen the validity of Opal’s activity and sleep measures on larger, more diverse populations, including those with neurological disorders.Our pilot study provided evidence for establishing concurrent validity of t Opal Actigraphy measures of sleep and physical activity with 3D accelerometer data during daily life, as compared to the most widely used ActiGraph. Historically, Opal multi-sensor solutions have enabled high resolution data capture of precise aspects of human mobility via prescribed movement tasks in controlled environments to assess the efficacy and safety of therapeutic interventions. The new and validated Opal Actigraphy single-sensor configuration can provide further insight on how movement impairments captured in clinic translate into real-world quality of life by quantifying overall physical activity levels and sleep durations. Future studies should evaluate Clario’s Opal Actigraphy solution in larger, more diverse cohorts and determine the clinically meaningful important difference in activity and sleep metrics in response to interventions.


This study presents findings on improvements to the Frontal and Sagittal Cobb angle, Global Spinal balance, and lung function parameters  in an adult male with idiopathic scoliosis suffering from pain during ADL and sports activities who was treated with a biomechanically designed exercise protocol.The 26-year-old male reported upper and middle back pain which worsened when playing cricket. Whole spine standing x-Ray AP view revealed a right thoracic Scoliosis (Lenke 1 curve) of Cobb angle 48.6° with left lumbar compensatory curve of 24.7°, Thoracic hypo kyphosis of 9.9°, and VAS rating for pain of 8. The patient was treated with myofascial release, stretching, aerobics, strengthening exercises, “Breathing with core” for stabilization, and biomechanically designed curve specific corrective exercises.Re-assessment 32 weeks post intervention demonstrated significant reduction in the major Cobb angle by 13.8°, minor Cobb angle by 9.5°, Thoracic hypokyphosis normalized to 37.8°, Coronal balance improved by 17.4 mm, Sagittal balance regained by 4.2 mm, Spine ROM improved by a total of 6.5 cm, Enhancement of pulmonary function of FEV1 by 7% and PEF by 18 litres/min, and dramatic improvement in aesthetics and pain perception.The biomechanically designed exercise protocol helped straighten the curve through curve specific corrective exercises and stabilized the curve by “Breathing with core”. It also treated the associated signs and symptoms of spinal pain syndrome by myofascial release and proper ergonomics, pulmonary dysfunction by aerobics, and muscle tightness and weakness  by stretching and strengthening. Scoliosis is a three-plane deformity of the spine, rib cage, and pelvis. Diagnosis is confirmed when the Cobb angle is 10° or more with recognizable vertebral axial rotations. After elimination of the other 20% known causes of scoliosis, when overall assessment and investigations fail to provide a definite reason for the development of the curve, it is diagnosed as Idiopathic Scoliosis. Among the three types, Adolescent Idiopathic Scoliosis is the most common and manifests and progresses during growth spurts in adolescence.A major percentage of patients diagnosed with Adolescent Idiopathic Scoliosis require conservative management, with the primary goal of restricting curve progression during growth and also reducing the curve. But many adult cases remain untreated due to lack of diagnosis in their adolescence and so need to be treated during adulthood. Curve progression during adulthood is documented and understood as an asymmetrical spine alters the biomechanical loading, causing the viscoelastic structures to undergo creep which initiates the structural bony changes and muscular deterioration. Therefore, bigger curves >30° are the victims of curve progression  as the cSo, exercises are in great need not only during growth and development but also during maturity and adulthood. A sound knowledge of spine biomechanics and the associated deformities is essential for comprehending and formulating exercise methods. It is also imperative to understand curve types, as the exercises are curve-specific.Among all classification systems, Lenke classification  is the mThe difference between lying and traction view reveals the degree of correctability of the concavity side soft tissues and the difference between standing and lying view indicates the degree of muscular inefficiency  which prIn April this year, a 26-year-old male presented to the Dept. of Orthopedics with complaints of back pain caused by his Right Thoracic Idiopathic Scoliosis (Lenke 1) diagnosed one month prior. He had visited other spine clinics before coming to NIMS Hyderabad, and was suggested for surgery. Turned off by the invasive procedure, he was instead seeking alternative management and was referred to NIMS. The patient did not present any ligament laxity and did not undergo any genetic testing.Family history disclosed consanguineous parents, but none of his siblings or cousins suffered from such a condition.Whole spine standing x-ray AP and Lateral and traction films of the patient were evaluated using the digital spine software tool Surgimap.During the initial assessment, the patient reported pain and tenderness in the right upper mid back, bilateral shoulder girdle, and anterior joint. Prolonged sitting produced low back pain and subsequent walking was troublesome with an aching sensation in the right (upper anterior aspect) and left thigh (upper posterior aspect), hypertonic muscle, and taut band along the curve convexity noted on palpation.Pain perception on a ten-point scale (VAS) was 8 on average.Thoracic Convexity side (Right)—Shoulder elevated, Posterior rotation of vertebral bodies palpable , Rib hump, Scapula protruded sitting on the rib hump and tilted anteriorly . Impingement and pain during arm elevation, unstable scapula during static and dynamic positions, Rotator cuff muscles felt inflamed and tender.The Angle of Inclination was 15° at the thoracic curve apex (Scoliometer measurement).Thoracic Concavity side (Left)—Posterior thorax appeared atrophied, anterior rotation of vertebral bodies palpable , Shoulder depressed downwards, Rotator cuff felt tender and painful.Lumbar convexity side findings—On inspection, the left buttock was more prominent.Observation from side—left pelvis posterior and right pelvis anterior (Pelvic rotation in transverse plane).On palpation, the left ASIS (Anterior superior Iliac spine) position was higher than the right ASIS, but the left PSIS (posterior superior Iliac spine) position was lower than the right counterpart .When the Innominate rotates anteriorly in the sagittal plane, the ASIS moves down and PSIS goes up, and vice versa. Here, the left Innominate rotated posteriorly and the right Innominate rotated anteriorly in the sagittal as well as transverse plane. This was visible in the pre-intervention radiograph as an asymmetrical width of both innominates, Lumbar concavity side findings—Waist indentation on right, Iliac crest moved up in the frontal plane to compensate for the left lumbar curve and maintain the coronal balance, making the right Quadratus Lumborum muscle tight.Xiphisternum to Medial Malleolus)- Left > right.by 2 cm, Standing true length - Right > Left by 2 cm, Standing length - No discrepancy.Standing Apparent limb length , Thoracic kyphosis = 9.9°, Lumbar lordosis = 54°.Negative Coronal balance = (−20.1) mm on standing AP radiograph, C7 offset as a measure of Coronal alignment , Positive Sagittal balance = (15.7) mm on standing lateral radiograph, (SVA) Sagittal vertical axis as a measure of Sagittal alignment, measured as the horizontal distance between C7 plumb line and Central Sacral vertical Line (CSVL).The predicted value of FEV1 = 4.01 liter, PEF = 568 L/m as per height, weight, gender, and age, (Measured on a smart one Spirometer).Achieved Value (FEV1) = 2.68 liter (67%), PEF = 550 L/m.After the initial assessment, the patient signed a consent form to participate in the study and undergo exercise treatment. The treatment intervention consisted of institutional-based treatment of two hourly sessions once a week and a home program of the same protocol twice a day on other days of the week for 8 months. Around 12 sessions were spent releasing the myofascial knots that restored the optimum resting tone of the hypertonic longissimus thoracis, enabling pain-free performance and effective learning of the exercises.curve specific correction in sitting with maximum effort for self-release and self-awareness of the corrected position . After habituation and improved tolerance to exercises, the frequency of each exercise was slowly increased to 10 with an increasing hold period. Subsequent to myofascial release, breathing with core and aerobics was performed for warming up. On completion of all exercises, breathing with core was performed once more for stabilization of the newly attained range.The patient was encouraged to attain the position repeatedly as in 1.Myofascial Release-Para spinal muscles on the convex side of the curve, Upper trapezius of right side, Rotator cuff of both side, Rectus femoris of lumbar concave side, hamstring of lumbar convex side.The above muscles were released using fingers, wrist pad, knuckles, elbows, and a manual massager.2.Breathing Exercises-(a)Valsalva breathing (b) Breathing with core(a)Valsalva Breathing- strengthens the diaphragm isometrically.(b)Breathing with core-This is the most important technique to strengthen the deep, local intervertebral muscles like Rotators, Intertransversarii, and Multifidus. In a recumbent position, the curve corrects to some extent, so the muscles are better aligned to develop optimal tension.This is carried out in a supine knee bent position, with arms by the side, and elbows kept at 90°. The subject squeezes the bowel and bladder to engage the pelvic floor muscles, elevating them upward, and presses the navel down to recruit the Transversus Abdominis. Both the feet and elbows press down towards floor to facilitate the core at the lower abdomen, chest, and neck level. While engaging these core muscles, the patient tries to breathe in and out. This pulls the Diaphragm origin down and facilitates Diaphragmatic excursions against the abdominal and pelvic pressure. The wave of contraction of all major core muscles engages the local deep muscles of the whole spine. This is better performed on an empty stomach for at least 15 min.3.Aerobics: (a) Yoga Cycling (b) Lunges to standing(a)Yoga Cycling.(b)Lunges to standing—Lumbar convex side leg forward, bringing the posteriorly driven innominate anterior and strengthening the quadriceps of this side, with the other leg backward in line with the body (5 min).4.Optimal Strengthening of Quadratus LumborumLying on the side with lumbar curve down, a towel roll kept underneath the curve to stretch the upper side Quadratus lumborum muscle, down leg flexed at hip and knee, upper leg lifted to a height just along the body line to strengthen the shortened muscle from an optimal position .5.StretchingUpper Trapezius—Self -stretch of right-side using the Muscle Energy Technique of Post Isometric relaxation.6.Optimal Strengthening of Scapular StabilizersThe prone arm is lifted in an 120° abduction and the Scapula tilted posteriorly by manual support for optimal torque development.The exercise protocol consisted of the following techniques:7.Rotation reversal Exercise for the Major thoracic CurveSubject sitting, thoracic convexity side arm kept against a wall with the shoulder at 90-degree abduction, elbow at 90-degree flexion, concavity side arm is also held in the same position, convex side arm pushes against the wall from the front side and concave side arm pushes to the back against therapist resistance. Simultaneous resisted contraction of horizontal arm adductor muscles from thoracic convex side facilitates anterior vertebral rotation and horizontal arm abductor from concave side facilitates posterior vertebral rotation that reverses the vertebral malrotation .8.Planks A side plank is held, keeping convexity side down, which stretches the upper concavity side muscles and strengthens the convexity side muscles.9.Strengthening of Trunk Flexors & Extensors-Performed in a neutral trunk position or quadruped position, head just clearing off the bed to avoid hyperflexion and hyperextension. The mechanical impact of scoliotic spine hyperflexion and hyperextension is explained below.During spinal flexion, vertebral bodies glide anteriorly over adjacent bodies  of convex side muscles against the subject's own arm resistance generated by pushing a small stick stood on the ground or pushing against any fixed surface . The patCorrective exercises in sitting are useful as the subject can sit in a corrective position in both his home and workplace. Bending ipsilaterally when sitting is a gravity-aided movement, and so is helpful for controlling creep on the convex side muscles .The patient sits on a small stool or arm-less chair, with the left leg kept in the front at the edge, and right leg kept as far back as possible so that the left side pelvis and lumbar bodies automatically come forward. The left foot is used to press any fixed surface from the lateral side to create active pressure for curve straightening. This sitting posture prevents back pain and helps prolonged sitting in a desk job.The patient bends to the convex side while the right trunk is kept forward and left trunk is held backward, performed similarly as when lying.—Lumbar and Pelvic correctionSubject stands on the right leg, kept back and adducted. The left foot taken forward pushes against a wall or any fixed surface from the lateral border  from the concave side towards the center, reducing the gravitational torque, while picking up objects, bringing the Right trunk to the front.Staircase walking-Lumbar convex side leg to progress first.Post intervention assessment revealed significant relief from all types of pain; the subjective VAS rating reduced from 8 to 1 and the patient could hold a new desk job which he had previously quit because of pain.The patient stated, “I am not getting any types of pain, and not worried about my future now as I know how to control this condition on my own”.There was a less protruded Scapula on the Right, left Scapula pronounced, and Symmetrical pelvic position , Angle of Inclination reduced to 10°.Correction of Apparent and True Limb length discrepancy by 1.5 cm.Thoracolumbar flexion and extension range enhancement by 3.5 and 3 cm respectively.Body weight reduced by 1 Kg with an improved body composition.Significant reduction of major Cobb angle by 13.8° from 48.6° to 34.8°, Minor Cobb by 9.5° from 24.7° to 15.2°.Thoracic Hypo Kyphosis of 9.9° normalized to 37.8°. Lumbar Lordosis of 54° improved to 69°.Negative Coronal balance of (−20.1) mm reduced to −2.7 mm.Positive Sagittal balance of 15.7 mm reduced to 11.5 mm.FEV1 of 2.68 liters (67%) changed to 3.00 liters (72%). PEF improved by 18 liters/min.Spinal pain syndrome in scoliosis can originate from different anatomical sites, such as annular and facet joint stress, neural tension, and fibrotic muscle, and physical therapy as a form of treatment is recommended by SOSORT and SRS to control this , 6.The varied regional pain of the present case can be explained on the basis of literature and clinical findings.The upper mid back pain is assumed to originate mainly from the fibrotic muscle along the convex side of the curve that got overburdened due to persistent work in the frontal plane to counteract the gravitational torque shift on the concave side. Stretch weakness also reduces the optimal tension producing abilities turning it hypertonic with chronic inflammation and fibrosis restricting motion and correction.An elevated shoulder and rib hump changes the orientation of the lower trapezius and Serratus anterior force couple, making the scapula unstable. Girdle and Rotator cuff pain could be due to a tight upper trapezius and overactive cuff muscle to stabilize the scapula, creating inflammatory changes.Shoulder depression on the thoracic concavity side submits the capsule and cuff to increased gravitational traction, forcing the cuff muscle to contract maximally to stabilize the head in the socket, making them inflamed and painful .distortion  increases tension in the right Hamstring, right Sacro tuberous ligament, and left Rectus Femoris muscle and decreases tension in the left hamstring, right Rectus Femoris muscle, and Iliotibial band  and stabilization of the attained range by strengthening of smaller, deeper local muscles and larger, superficial, global antigravity muscle.The superficial hypertonic longissimus thoracis inhibits the deeper local stabilizers that help brace the spine internally . SubsequImprovement in other morphological parameters, like posture and aesthetics, and functional parameters like ROM, flexibility, body mass composition, pulmonary function measures, normalization of thoracic kyphosis, and sagittal and coronal balance  are advaThe limitation of the treatment method is that the exercises cannot be performed without institution-based therapy sessions in the beginning, as they are needed to release myofascial adhesions and effectively learn the corrective techniques. Once the patient masters self -release with self- awareness and breathing techniques with all corrective exercises, they can be performed anywhere with continued improvement over time.The limitation of the case is that this improvement is shown in only a single case.This case report proves the plausibility of curve reduction in adult cases with a biomechanically designed exercise protocol. The protocol can correct the multiple signs and symptoms of idiopathic scoliosis, reverse all dimensions of deformity, control spinal pain syndrome of scoliosis, and provide a sustainable correction. Irrespective of variable etiological factors behind the initiation of Idiopathic Scoliosis, the musculoskeletal dysfunction sequelae are similar, and they can be treated against this background. The corrective exercises are comprehensive, curve-specific, and feasible to be applied for all Lenke curve types by changing the direction to a suitable position as per the needs of the patient.More time is required to assess the level of improvement beyond a 1-year period of regular adherence to the protocol.


Despite significant efforts made to control malaria in Ethiopia, the disease remains one of the top public health problems in the country. Baseline malaria prevalence and associated factor at high malaria area is important to guide malaria control interventions, there was paucity of information regarding the study area. Therefore, the aim of this study was to determine prevalence of malaria and associated factors among febrile adults in Siraro district health facilities, West Arsi Zone, Oromia, Ethiopia.Institution-based cross-sectional study was conducted among 317 febrile adult patients at Siraro district health facilities. Structured pre-tested questionnaires were used to collect data. Epi-data version 3.1 and SPSS version 23 were used for data entry and analysis respectively. In order to identify factors associated with malaria infection bivariable and multivariable binary logistic regression analysis was employed, The Adjusted Odds Ratio (AOR) with a 95% confidence interval (CI) and p-value of < 0.05 was computed to show the strength of the association.The overall prevalence of malaria at the study area was 130 (41.0%) [(95% CI 35.3–46.7)]. Occupation (being farmer) , having poor knowledge on malaria transmission , house with wood wall , and number of windows (≥ 3) in the house  were identified to be significantly associated with magnitude of malaria in the study area.The prevalence of malaria at the study area was high as compared with the national wide figures. Being farmer, having poor knowledge on malaria transmission, and housing condition  were found to be significantly associated with malaria infection in the study area. Therefore, there has to be an emphasis on addressing the factors by providing sustainable health education for the communities to improve their housing condition and knowledge of community on the way of malaria prevention. Plasmodium parasites, mainly Plasmodium falciparum and Plasmodium vivax, that are transmitted by the bite of infected female Anopheles mosquitoes [Plasmodium falciparum is the most prevalent malaria parasite in the World Health Organization (WHO) African Region, accounting for 99.7% of cases, whereas P. vivax is the predominant parasite in the WHO Region of the Americas, representing 75% of malaria cases in 2018 [P. falciparum malaria can progress to severe illness and death within a period of 24 h [Malaria is a life threatening, but easily preventable and treatable a vector-borne disease that continues to have a devastating impact on the health and livelihood of people around the world . Malariasquitoes . Plasmod of 24 h .Malaria is one of the most severe public health problems worldwide and identified as a leading cause of death and disease in many developing countries . Nearly P. falciparum and 30.8% were P. vivax [Anopheles gambiae complex, Plasmodium falciparum, local weather conditions often allow transmission to occur year round, scarce resources and socio-economic instability, parasite and vector control measures, and knowledge, attitudes and malaria prevention practices [Globally in 2020, there were an estimated 241 million cases and 627,000 death of malaria . This reractices .. Even though the area categorized as high malarias area no study found about the magnitude and risk factor associated with malaria. Therefore, this study aimed to understand the burden and risk factors associated with malaria, which would help the government and stakeholders to plan strategies for malaria prevention and control.The global scale-up of malaria prevention interventions has saved millions of lives globally and cut malaria mortality by 36% from 2010 to 2020, leading to hopes and plans for elimination and ultimately eradication. However, the magnitude is still remains to be stagnant . MoreoveInstitutional-based cross-sectional study design was conducted from June 25 to July 20/2022.At public health facilities providing malaria treatment services at Siraro Woreda, Oromia Regional State, South Ethiopia. Siraro Woreda is located about 56 km to West of Shashamane city, capital of West Arsi Zone, and about 330 km from Addis Ababa capital city of Ethiopia. In the Woreda there are 6 rural clusters and 32 kebeles with total population of 207,541 . Almost all areas of the Woreda are cash crop areas; the main crops cultivated in the Woreda are boloke and maize. A number of public and private health facilities are providing health care services for those populations. Public health facilities are one Primary Hospital, 6 health centres and 28 health posts. In addition to these, there are six private health facilities.(n= (Zα/2)2 *p (1 − p)/d2) by considering the following assumptions: prevalence of malaria in East Showa, Ethiopia, which was 25% [= 317.All febrile adults under the catchment area and randomly selected febrile adult patients who visited Siraro district public health facilities during the study period were the source and study population of our study. Sample size for the first objective was determined using a formula for a single population proportion  was 25% , d = 5% Sample size for the second objective, which is risk factor malaria, is calculated using double population proportion formula taking more frequently observed associated factors for risk factor for malaria  , 22 usinThe data were collect by a pretested structured interviewer-administered questionnaire, which were adapted from previous peer-reviewed literature –10, 13. Anopheles mosquito transmit malaria, (5) feeling cold and rigor is sign of malaria, (6) fever is a sign of malaria, (7) malaria is curable disease, and (8) use of ITNs prevents malaria infection. Attitude of the participants was evaluated by eight items: (1) think anyone can get malaria; (2) malaria is a serious and life-threatening  disease, (3) the best way to prevent myself getting malaria is to avoid getting mosquito bites, (4) sleeping under a mosquito net during the night is one way to prevent myself getting malaria, (5) can be risky when malaria medicine is not taken properly and completely, (6) need to visit health centre/clinic when I am suspected of malaria, (7) think one can prevent him/herself from getting malaria, (8) malaria can be transmitted from person to person.The participants’ level of knowledge were assess by eight items: (1) malaria is preventable, (2) stagnant water is mosquito breeding site, (3) mosquito biting time is at night, (4) female Practice of malaria prevention was evaluated by 10 questions about (1) How often do you visit the health center/clinic when you fall sick, (2) How do you describe your habit of visiting health centre/clinic when any other family member falls sick, (3) How often do you sleep in an insecticide-treated mosquito net (ITN), (4) How often do other family members sleep in mosquito nets, (5) How do you describe your habit of checking for holes/repair mosquito nets, (6) How often do you use anti-mosquito spray (IRS) in your house, (7) How do you describe your habit of trimming bushes around your home.Three trained nurses and two BSc holders Health Officers working in Siraro district Health centres were participate as the data collectors and supervisors, respectively. Two days of training had bestowed for both data collectors and supervisors with regard to the importance of the study, how to keep privacy, and the confidentiality of the study participants. Moreover, 5% (n = 16) of questionnaires tested to nearby district  health facility. Fortunately, there was no adjustment was done. The principal investigator together with supervisor checked and reviewed questionnaires on daily bases to ensure the completeness and consistency of information collected. The collected data coded, and entered using Epi-Data version 3.1 to maintain the quality of the data.The Collected data entered by Epi-Data version 3.1 and exported to SPSS version 23 for data analysis. Descriptive and analytical statistical methods used to describe and infer the findings respectively. Tables, figures, and text used to present results. Binary logistic regression analyses run to assess the statistical association between malaria prevalence and each independent variable. Accordingly variables that was statistically significant at p-value of < 0.25 during bivariable binary logistic regression were entered into multivariable binary logistic regression to control the role of confounding variables. Multicollinearity was tested by using the variance inflation factor (< 10). A reliability analysis of the questionnaires was checked and Cronbach’s alpha showed the questionnaire were passed the acceptable reliability number (α = 0.87). The Hosmer–Lemeshow test used to assess the model fitness. Adjusted odds ratios (AOR) with 95% CI estimated to assess the strength of associations and statistical significance declared at a p-value < 0.05.Eight items used to assess the participants’ level of knowledge. Each item was assessed with a 2-point scale having a score of 0 = for each incorrect answer (No), and 1 = for each correct answer (Yes). Then the final score for each scale calculated by adding up the points obtained for the corresponding items by SPSS compute command. Accordingly, the overall score range from a minimum of 0 to a maximum of 8 points. Hence, those participants who answered above the mean score (≥ 5.44) regarded as having a good knowledge. Whereas participants who answered less than the mean score (< 5.44) of the questions were taken as having poor knowledge .To determine the attitude of the participants toward malaria, 8 items were used. Each item was assessed with a 5-point Likert scale with scores ranging 1–5 . The overall score range from a minimum of 8 to a maximum of 40. Thus, participants who answered above the mean score (≥ 22.05) regarded as having a positive/favourable attitude. Whereas participants who answered less than the mean score (< 22.05) of the questions were taken as having negative/unfavorable attitude .To determine the practice of the participants toward malaria prevention, seven items were used. Each item was assessed with a 3-point Likert scale with scores ranging 1–3 . The overall score range from a minimum of 7 to a maximum of 21. Thus, participants who answered above the mean score (≥ 12.35) regarded as having good practice. Whereas participants who answered less than the mean score (< 12.35) of the questions were taken as having poor practice .Three hundred seventeen 317) respondents were participated in the study with the response rate of 100%. The mean (± SD) age of the respondents was 34.73 (± 11.75) years. Majority of study subjects, 190 (59.94%) and 144 (45.43%) were females and Oromo ethnics respectively. Among the vast majority of respondents 220 69.4% of them were married. Above half of the study subjects 219 (69.1%) were rural resident. The majority educational status 135 (42.6%) was secondary followed by primary 89 (28.1%) (Table 7 respondOf 317 study participants’ 72 (22.71%) houses were tacked (attached) and 157 (49.53%) houses wall were constructed from wood. Among 317 respondents 189 (59.62%) of them responded that there is open and stagnant water around their residency. Of 317 study subjects 95 (29.97%) of them has no ITNS in their house and from those ITNS ownership 71 (22.40%) of them has < 2 ITNS (Table Among 317 respondents’ majorities 245 (77.3%) of them replied that; Malaria is preventable disease and 136 (42.9%) respond that; stagnant water is not mosquito breeding site. Of 317 study participants 228 (71.9%), 244 (77%) of them respond that; feeling cold/rigor and fever is sign of malaria respectively. The overall good knowledge status of the study participants was 162 (51%) (Table Of 317 febrile adult patients at Siraro district 82 (25.9%) and 69 (21.8%) of them disagree and strongly disagree, respectively, on malaria is a serious and life-threatening  disease. From a total study subjects about; 89 (28.1%) of them were agreed while 78 24.6%) of them were disagreed on avoid mosquito breeding site (stagnant water) prevent malaria. Majorities of study participants 85 (26.8%) greed that sleeping under a mosquito net during the night is way of malaria prevention  (95% CI 35.3–46.7)  , having poor knowledge on malaria transmission , housing wall material (wood) , and number of windows (≥ 3)  were significantly associated with magnitude of malaria in the study area.Accordingly, the odds of malaria prevalence were 6 times higher among famers as compared to merchants . Participants who have poor knowledge on malaria transmission were nearly three times higher odds of contracting malaria as compared to those participants who have good knowledge . Participants whose house wall constructed form wood were nearly three times more likely to develop malaria as compared to participants whose housing wall constructed from cement . On the other hand, peoples living in the houses with three and above windows were 6.82 times  more likely infected by malaria as compared to peoples living with one window house  for the years 2014–2020 had anticipated that by 2020 all households living in malaria endemic areas would have the knowledge, attitudes and practice towards malaria prevention and control, to reduce malaria cases by 75% from baseline of 2013 and to eliminate malaria in selected low transmission areas in Ethiopia . Thus, tThe prevalence of malaria in this study is consistent with a systematic review and meta-analysis done in Ethiopia (37.6%) based on data obtained from studies conducted on mixed regions of low lands . HoweverAgriculture and malaria has intertwined, a study found that irrigated rice-growing communities are exposed to more mosquitoes and higher malaria risk . This stParticipants who have poor knowledge on malaria transmission methods were nearly three times more likely of being infecting with malaria as compared to participants who have good knowledge. The finding has supported by study done at southwestern coastal region of India , Malawi In this study, the housing condition has found to be significantly associated with the magnitude of malaria infection among the study participants in Siraro district. Study participants who lives in the house with wood wall were more likely to have malaria infection than participants who were lives in the house with cement wall. Likewise, peoples living in the houses with more than three windows were higher likely of infecting by malaria as compared to peoples living with one window house. The finding is in line with study conducted in Eritrea where malaria infection is mostly common among peoples living in mud wall houses . SimilarThe prevalence of malaria at the study area was high as compared with the national wide figures. Factors like; occupational status being farmer, having poor knowledge on malaria transmission, housing condition  were found to be significantly associated with the observed magnitude of malaria in the study area. Therefore, there have to be given an emphasis on addressing the root causes of the problem via providing sustainable health education for the communities to improve their housing condition. Siraro district health office and health facilities in collaboration with stakeholders should have to work on malaria prevention strategy to reduce this high burden of malaria cases in the district.