It is also possible that some of the "vesicles" may represent red cell fragments rather than "microvesicles" and it may be considered technically challenging to separate the two populations by flow cytometry alone.

However, majority of these fragments as seen on a blood film are usually in the size range of 1 μm or above, and in the range of the size of large platelets.

12 In previous studies evaluating the detection of fragmented red cells, using an automated hematology analyzer, have also used the principle of light scatter.

The average dimension of the fragments was about 30 fL with a unidimensional size of a few microns when compared to a red cell which ranges between 80 to 100 fL with a diameter of about 8 μm.

13,14 The gating strategy used in our exper- iments excluded events larger than 1 μm in the MV gate therefore ensuring only red cell MV were enumerated.


The reason for apparent low levels of circulating endothelial MV could be due to changes in the size or surface antigen expression of the MV which may be altered in response to envenomation, resulting in MV that are undetectable via our method of flow cytometry.

This was an unexpected finding that requires further investigation.


Limitations of this study include that we did not perform any functional studies in MV isolates or use alternative methods to eval- uate MV apart from flow cytometry.

Inter- laboratory as well as inter- instrument variability in flow cytometry is a concern despite the fact that it is an easily and widely available technique.

Also, MV smaller than 200 nm are usually not detectable by flow cytometry.

The use of isotype controls, correlation with functional testing and participa- tion in global standardization initiatives may reduce this variability.

15 Given the small sample size, we did not stratify the results according to gender or comorbidity status but this may be important to explore in a larger cohort.


There may be some variability in how snake venom interacts with the human coagulation system.

However, all of the snake venoms in the current study are well known to contain procoagulant toxins that closely resemble part or all of the mammalian prothrombinase complex and produce identical clinical sequelae.

The toxin in brown snake is the most potent, being most similar to the FXa- Va com- plex, and able to directly cleave prothrombin to produce activated thrombin.

The procoagulant toxin in tiger snake resembles FXa and requires completion of the complex from human plasma FVa to en- able thrombin activation.

1 In summary, we provide the first report of changes in endovas- cular MV in snakebite patients and their possible role in the patho- physiology of snakebite- related MAHA.

This study provides proof of principle that MV levels could be a marker for direct toxin mediated damage to red cells, platelets, and endothelial cells, that has been hitherto difficult to define.

We established that flow cytometric as- sessment of MV could be used as an important technique for mea- surement of direct toxin mediated damage to red cells in snakebite associated MAHA.


AKE is a recipient of NSW Health Pathology/HCRA/CMN trans- lational research fellowship.

This study was partly supported by Calvary Mater Research funds and NSW Health Pathology-Hunter Trust Funds.

GKI is supported by a NHMRC Senior Research Fellowship ID1061041 and the Australian Snakebite Project is par- tially funded by an NHMRC Centre for Research Excellence Grant ID1110343.

We acknowledge the various hospitals who participated in ASP study and provided the samples as well as the study patholo- gist who provided review of blood films.

There are no conflicts of interest for any of the authors.


Snakebite envenoming is estimated to cause 95,000-125,000 deaths and up to 400,000 permanent injuries or disabilities worldwide on an annual basis.

This serious health threat has been recognized by the World Health Organization, and snakebite envenoming is classified as a category A neglected tropical disease [1].

In May of 2018, the World Health Assembly adopted a resolution on snakebite envenoming [2], thereby providing a mandate to develop a comprehensive plan to effectively control the burden of envenoming and reduce its debilitating impact.

The multisectoral strategy that is required to achieve this will address socioeconomic, environmental, and healthcare challenges.

Targeted actions to raise awareness have culminated in, among others, a recent international scientific conference where many essential stakeholders united in the fight against snakebite envenoming [3].


While the highest health burden from venomous snakebites falls on south Asia and sub-Saharan Africa [4], exact information on the number of envenomings, clinical effects of snakebites, and effective treatments are lacking on a global scale.

In an effort to address this, the Australian Snakebite Project registry has compiled information on a substantial proportion of all Australian snakebites since 2005 [5, 6].

This study clarified that the most commonly observed clinical envenoming phenotype in Australia is venom-induced consumptive coagulopathy (VICC) [5], an unbridled activation and consumption of the coagulation system.

This is due to the venom composition of the snakes that are most commonly involved in these envenomings [5]; Pseudonaja species contain procoagulant factor Xa-Va-like and Notechis species factor Xa-like enzymes [7-9].

Once injected into the blood stream of the prey, the Notechis factor Xa-like protease interacts with prey factor V(a) [10], and the circulating venom factor Xa-Va-like enzymes are capable of converting prothrombin of the prey into thrombin [10, 11].

Action of thrombin induces VICC, which is characterized by a (complete) consumption of fibrinogen and factors V and VIII, elevated D-dimer levels, and an international normalized ratio >3 [12, 13].


From the envenomed individuals that developed VICC as reported in the Australian Snakebite Project, approximately 10% presented with microangiopathic hemolytic anemia (MAHA) [5], resulting from intravascular red cell fragmentation with schistocytosis and thrombocytopenia.

Most MAHA cases were observed following taipan (Oxyuranus species, 26% MAHA cases) and brown snake (Pseudonaja species, 14% MAHA cases) envenoming, while bites from Notechis and Hoplocephalus species each led to MAHA in 7% of envenomed individuals.

Interestingly, this correlates with the venom composition of these snakes, with both Oxyuranus and Pseudonaja venom comprising factor Xa-Va-like prothrombin activators, while a factor Xa-like protease is found in the venom of Notechis and Hoplocephalus snakes [7-9].

Also, the onset of VICC appears to proceed more rapidly following envenoming by Pseudonaja snake bites relative to those of Notechis snakes [14], and MAHA only seems to occur following VICC-associated snakebites [5].

Despite these indirect observations, the exact pathophysiology of venom-associated MAHA remains unclear.


To gain insight into the venom-mediated damage to the endovascular system, in their recent article in Research and Practice in Thrombosis and Haemostasis, Enjeti and colleagues have focused on the role of microvesicles (MVs) in snakebite patients [15].

Microvesicles are shed by outward blebbing of the plasma membrane and typically function to transfer cargo, such as proteins and ribonucleic acids, from a donor cell to an acceptor cell, leading to behavioral changes in the acceptor cell.

Microvesicles may be shed by platelets, endothelial cells, megakaryocytes, and tumor cells, and the diameter of these vesicles ranges from 30 to 1000 nm [16, 17].

Elevated levels of MVs in the circulation are often associated with pathophysiological conditions such as sepsis, diabetes mellitus, cardiovascular diseases, and cancer [18-22], and MVs may even impact progression of disease.

In their study, Enjeti and colleagues primarily studied MVs generated by platelets, red blood cells, and endothelial cells.


Compared to control subjects, platelet MVs were significantly higher in the blood of snakebite patients that did not suffer from MAHA, while paradoxically red blood cell–derived MV levels were enhanced in patients experiencing MAHA [15].

In snakebite patients either suffering from MAHA or not, endothelial MVs were decreased.

These observations led the authors to speculate on the usefulness of red blood cell MVs as a surrogate marker for MAHA.

While the results of the current study are certainly of interest, a number of drawbacks may be identified.


First of all, characterization of the MVs in this study could have been more extensive; flow cytometry was applied to detect MVs, and this method only detects larger MVs.

Thus, the majority of MVs (with a diameter of <400 nmol L−1) may have been missed.

It remains unclear how many of the red blood cell MVs were actually red cell fragments, with fragmentation being a hallmark of MAHA.

Second, while the diagnostic use of MVs in snakebite patients sounds attractive, equipment allowing for high throughput analysis of MVs in snakebite patients is not readily available.

More rapid and precise diagnostic strategies to identify envenomed patients with MAHA could lead to a faster administration of specific treatment modalities.


Current therapy of envenomed patients consists of specific antivenom administration, with early treatment (preferably within the hour post-snakebite) being most effective, as VICC develops very rapidly [14].

However, VICC has also been reported to resolve within 24-48 hours without treatment [10], which is likely due to resynthesis of the depleted coagulation proteins.

While the reports are scarce with respect to the treatment of MAHA, this syndrome seems to resolve following toxin neutralization by antivenom administration [10].


Finally, the current study failed to answer why envenomation leads to MV generation and if these MVs play a role in MAHA and/or the coagulopathy often seen in snakebite patients.

Furthermore, it is ambiguous whether MAHA is secondary to VICC, and if so, whether fibrin deposition in the microvasculature could contribute to the pathophysiology of MAHA.

While this would be consistent with the formation of schistocytes and the observed thrombocytopenia, the correlation with both platelet and endothelial MVs remains to be identified.

Until further work is performed on the link between MVs and venom-associated MAHA in snakebite patients, a causal role for such MVs in these pathological processes may just be a popping bubble.


We thank the authors Bos and Veersteg for their insightful commentary [1] on our brief report entitled “Circulating Microvesicles in Snakebite Patients with Microangiopathy” recently published in Research and Practice in Thrombosis and Haemostasis [2].

We echo their concern that venom-induced consumption coagulopathy (VICC) is one of the most medically important envenoming syndromes worldwide, particularly in tropical areas [3].

The activation of the clotting pathway by procoagulant venom toxins produced by many Viperid snakes and certain elapids, including Australian elapids, results in consumption of clotting factors leading to high risk of major hemorrhage in the victims [4].

However, the occurrence of VICC and microangiopathic hemolysis (MAHA) is an unintended complication in human envenoming.

In animals, usually meant to be prey for snakes, venom post-synaptic neurotoxins play a more central role by paralyzing the animal [5].

Hence, in vivo animal models for coagulopathy have been difficult to establish because of differential effect of snake venom on different animal plasma [5].


MAHA occurs in a smaller proportion of snake bite victims with VICC and is often associated with thrombocytopenia/anemia [6].

The mechanisms of endothelial damage and precise risk for developing MAHA are poorly understood.

The results from our study provide, for the first time, the investigation of platelet, red cell, and endothelial-derived microvesicles (MV) as measured by flow cytometry in the plasma of snake bite patients recruited from the Australian Snakebite Project (ASP).

Essentially, in this hypothesis-generating study, it was observed that red cell MV are significantly higher in snake bite patients with VICC and MAHA when compared to snake bite patients with VICC and no MAHA (Figure 1).

The mechanisms for production of MV and red cell fragments are likely very different.

We believe that these 200- to 1000-nm MV are distinct from larger red cell fragments of a few microns that have been described from studies using the automated hematology analyzers [7, 8].


More importantly, a markedly elevated red cell MV level detected by flow cytometry may be used as a surrogate marker for MAHA whereas thrombocytopenia alone may not be able to discriminate between those snakebite patients with evolving MAHA and those without.

In spite of the limitations in using flow cytometry, it is easily available and its use in routine diagnostics is widespread.

In fact, it is one of the few techniques extensively investigated for measurement of MV and where standardization recommendations have been published from across three workshops by the International Society on Thrombosis and Haemostasis [9-11].

The MV detected by flow cytometry have also been described to be more relevant for coagulant function in human plasma [12].

As technology advances, the availability of bench-top hematology analyzers with flow-cytometric capacity in many tropical reference centers is not unforeseeable in the near future.


While our results do not resolve the mechanistic basis of MAHA, the finding of low endothelial MV in snake bite victims is intriguing.

In vitro models using endothelial cell culture exposed to snake venom could be one approach to understanding this effect.

Measuring leucocyte-derived MV would be useful in considering the role of snake venom–induced leukocyte damage and its contribution to the coagulopathic process.

Serial sampling could provide valuable information on the time course of the pathophysiological events.

We also envisage that functional studies would add value to measurement of the red cell, endothelial, and platelet “bubbles” (ie, MV) after envenoming as a segue to understanding the “trouble” (ie, pathophysiological process) underpinning VICC and MAHA.


GenBank (1) is a public database of all known nucleotide and protein sequences with supporting bibliographic and biological annotation, built and distributed by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine (NLM), located on the campus of the US National Institutes of Health (NIH).


NCBI builds GenBank primarily from the direct submission of sequence data from authors.

Another major source of data is bulk submission of EST and other high-throughput data from sequencing centers.

The US Office of Patents and Trademarks (USPTO) also contributes sequence data from issued patents.

The data are supplemented by sequences submitted to other public databases.

Through a long-standing international collaboration with the EMBL Data Library (2) in the UK and the DNA Databank of Japan (DDBJ) (3), data are exchanged daily to ensure that all three sites maintain a comprehensive collection of sequence information.

NCBI makes the data available at no cost over the Internet, by FTP access and by Web text and sequence similarity search services.

NCBI also offers a wide range of WWW retrieval and analysis services which operate on the GenBank data (4).


GenBank continues to grow at an exponential rate.

Over the past 12 months 2.1 million new sequences have been added.

As of Release 113 in August 1999, GenBank contained over 3.4 billion nucleotide bases from 4.6 million different sequences.


Complete genomes (http://www.ncbi.nlm.nih.gov/Entrez/Genome/ org.html ) represent a growing portion of the database, with 14 of the 22 complete genomes now in GenBank deposited over the past 2 years.

Recent additions include Aeropyrum pernix K1, the deep sea archaea bacterium, Pyrococcus abyssi and the nearly complete genome of Caenorhabditis elegans.

There are at least 40 additional microorganism genomes, plus that of Drosophila melanogaster, that are being sequenced.

Many of these are expected to be in the public databases over the coming year.


Historically, GenBank had been doubling in size about every 18 months, but that rate has accelerated to doubling every 15 months due primarily to the enormous growth in data from expressed sequence tags (ESTs).

Over 63% of the sequences in the current GenBank release are ESTs, and current EST projects for human, mouse, rat and other organisms will contribute still more data.


Over 55 000 different species are represented in GenBank and new species are being added at the rate of 1250 per month.

Human sequences constitute 56% of the total sequences (34% of all sequences are human ESTs).

After Homo sapiens, the top species in GenBank in terms of the number of bases include Mus musculus, C.elegans, D.melanogaster and Arabidopsis thaliana.

Database sequences are processed and can be queried using a comprehensive sequence-based taxonomy (http://www. ncbi.nlm.nih.gov/Taxonomy/taxonomyhome.html ) developed by NCBI in collaboration with EMBL and DDBJ and with the valuable assistance of external advisors and curators.

The NCBI taxonomy is also covered in a separate article in this issue (4).


Each GenBank entry includes a concise description of the sequence, the scientific name and taxonomy of the source Nucleic Acids Research, 2000, Vol. 28, No. 1 organism, bibliographic references, and a table of features (http://www.ncbi.nlm.nih.gov/collab/FT/index.html ) that identifies coding regions and other sites of biological significance, such as transcription units, repeat regions, sites of mutations or modifications and other sequence features.

Protein translations for coding regions are also in the feature table.


The files in the GenBank distribution have traditionally been divided into 'divisions' that roughly correspond to taxonomic divisions, e.g., bacteria, viruses, primates and rodents.

In recent years divisions have been added as needed for specific initiatives in biology, such as divisions for EST sequences, genome survey sequences and high throughput genomic sequences.

There are currently 16 divisions.

For convenience in file transfer, the larger divisions, e.g., EST and primate, are divided into multiple files when posting the bimonthly GenBank releases on NCBI's FTP site.


ESTs continue to be the major source of new sequence records and genes.

Last year there were 1 765 860 sequences in the EST division of GenBank.

Over the past year the number of ESTs has increased by >70% to the current total of 3 017 997 sequences representing over 180 different organisms.

The top five organisms include: human, with 1 587 029 sequences (53% of the total); mouse, with 696 281 sequences (23%); rat, with 124 350 sequences (4%); nematode, with 100 844 sequences (3%); and the fruit fly, with 86 121 sequences (3%).


ESTs also continue to provide the major source of new gene discoveries.

As part of its daily processing of EST data, NCBI identifies through BLAST searches all homologies for new EST sequences and incorporates that information into the companion dbEST database (5).

In order to organize the EST data in a useful fashion, NCBI maintains the UniGene (http:// www.ncbi.nlm.nih.gov/UniGene/ ) collection of unique human (6), mouse and rat genes.

Additional information about UniGene is included in a separate article in this issue (4).


The STS division of GenBank currently contains >83 000 sequences and includes anonymous STSs based on genomic sequence as well gene-based STSs derived from the 3 ends of genes and ESTs.

These STS records usually include primer sequences, annotations and PCR reaction conditions.


The ultimate purpose for creating high resolution physical maps of the human genome is to create a scaffold for organizing large scale sequencing (7).

Physical maps based on STS landmarks are used to develop so-called 'sequence-ready' clones consisting of overlapping cosmids or BACs.

As the HTG sequence data derived from these clones are submitted to GenBank, STSs become crucial reference points for organizing, presenting and searching the data.

NCBI uses 'electronic PCR' to compare all human sequences with the contents of the STS division of GenBank; this identifies primer-binding sites on the human sequences that may be amplified in a PCR reaction.

This tool permits the assignment of an initial location on the map for sequence data and the association of existing GenBank entries to the new reference sequence.

The electronic PCR tool is also being made publicly available on the Web to enable any researcher with a new human sequence to relate that sequence to existing maps and HTG sequence data.


The Genome Survey Sequences (GSS) division of GenBank has been the fastest growing division in the last year, having increased 4.5-fold to a total of 1 008 904 records with 518 917 227 nucleotides.

GSS records represent 'random' genomic sequences, but are predominantly represented by 'BAC ends' which are single reads from bacterial artificial chromosomes used in a variety of genome sequencing projects, notably that of human (819 918 records), Oryza sativa (49 585) and Fugu rubripes (32 030 records).

The human data is being used (http:// www.ncbi.nlm.nih.gov/genome/clone ) along with the STS records in tiling the BACs used for the Human Genome Project (8).


The high throughput genomic sequences in the HTG division of GenBank are unfinished large-scale genomic records that are in transition to a finished state, after which they will be placed in the appropriate organism division (9).

These records are designated as Phase 0-3 depending on the quality of the data.

Phase 0 records consist of survey sequences generated to characterize clones and may or may not progress to Phase 1.

Phase 1 records contain unfinished sequence, and may consist of unordered, unoriented contigs with gaps.

Phase 2 records contain unfinished sequence as ordered, oriented contigs, with or without gaps.

Phase 3 records consist of finished sequence, with no gaps and may have annotations.

When a HTG record reaches phase 3 it is moved from the HTG division into the appropriate organismic division of GenBank.


It is now clear that a great number of human sequences will remain in the unfinished (HTG) division of GenBank as working draft sequence, while completed sequences will continue to move to the corresponding organismic division (PRI).

Together these two divisions should add some 2000 Mb of new genomic sequences from US-sponsored laboratories within the next year.


Each GenBank DNA sequence record is assigned an accession number, which is a stable and unique identifier for the GenBank entry as a whole, and does not change, even when there is a change to the sequence or annotation.

In order to identify specific sequences from different sources, as well as keep track of modifications to the actual sequence data, NCBI additionally assigns a unique identifier, termed a 'gi' number, to each sequence.

When a change in a sequence occurs, a new gi number is assigned to the new sequence version.

These gi numbers appear in the 'NID' (Nucleotide ID) field of a GenBank record, immediately following the ACCESSION field.


By agreement among the collaborative DNA sequence databases, a third identifier was introduced in February 1999 which consolidates the information present in both the gi and accession numbers.

GenBank displays this identifier on the VERSION line, which appears below the NID line in the GenBank flat-file format and is of the form 'Accession.version'.

For example, an entry appearing in the database for the first time has a VERSION number equivalent to the ACCESSION number followed by '.1' to reflect that this is the first version of the sequence in this entry, e.g., The VERSION line also displays the gi number.

If the nucleotide sequence changes, then so will the gi number and the version, but the accession will remain the same.

Although the NID line carries redundant information, this line remains in the file to ensure compatibility with existing programs.


A similar system for tracking changes in the corresponding protein translations was also introduced in February 1999.

Protein sequences now have identification numbers (in the format of three letters followed by five digits, e.g., AAA00001) that do not change, followed by a version number that increases with each subsequent version of the sequence.

This identifier appears as a qualifier for a CDS feature in the FEATURES table portion of a GenBank entry, e.g., /protein_id='AAA00001.1'


Protein sequence translations also currently receive their own unique gi number, which appears as a second qualifier on the CDS feature: /db_xref='PID:g1234567'.

The letter prefix indicates the database of origin for these identifiers (d=DDBJ, e=EMBL, g=GenBank).

Eventually, the gi number will be phased out since the new 'protein_id', complete with the version number, will represent both a unique identifier and a means to identify changes in the sequence.


The data in GenBank, and the collaborating databases EMBL and DDBJ, come from two sources: (i) individual authors who submit data directly to one of the databases, and (ii) bulk submissions from sequencing centers in the form of ESTs, STSs, GSSs or large genomic records (usually sequences from cosmids, BACs or YACs).

Data are exchanged daily with DDBJ and EMBL so that the daily updates from NCBI servers incorporate the most recently available sequence data from all sources.


Virtually all records enter GenBank as direct electronic submissions, with the majority of authors using the BankIt or Sequin programs.

Many journals require authors with sequence data to submit the data to a public database as a condition of publication.


GenBank staff can usually assign an accession number to a sequence submission within 2 working days of receipt, and do so at a rate of several hundred per day.

The accession number serves as confirmation that the sequence has been submitted and allows readers of the article to retrieve the relevant data.


All direct submissions receive a systematic quality assurance review including checking for vector contamination, verifying proper translation of coding regions, and checking for correct taxonomy and bibliographic citations.

A draft of the GenBank record is passed back to the author for review before it enters the database.


Authors have the right to request that their sequences be kept confidential until the time of publication.

In these cases, authors are instructed to inform GenBank staff of the publication date of the article in which the sequence is cited in order to ensure a timely release of the data.

GenBank policy requires that deposited sequence data be made public when the sequence or accession number is published.


Although only the submitting scientist is permitted to modify sequence data or annotations, all users are encouraged to report lags in releasing data or possible errors or omissions to GenBank at update@ncbi.

nlm.nih.gov


Several large-scale sequencing projects are producing megabases of human genomic DNA sequence.

NCBI works closely with sequencing centers to ensure timely incorporation of these data into GenBank for public release.

In parallel, NCBI has developed methods to integrate these sequences with genetic and physical map data and to search the sequences more effectively (e.g., through options in BLAST to mask Alu and other types of repetitive elements).

GenBank offers special batch procedures for large-scale sequencing groups to facilitate data submission, including the program 'fa2htgs' and other tools (10).


About 40% of individual submissions are received through a Web-based data submission tool, BankIt (http://www.ncbi.

nlm.nih.gov/BankIt ).

With BankIt, authors enter sequence information directly into a form, edit as necessary and add biological annotation (e.g., coding regions, mRNA features).

Free-form text boxes allow the submitter to further describe the sequence, without having to learn formatting rules or use restricted vocabularies.

BankIt creates a draft record in GenBank flat-file format for the user to review and revise.

BankIt is the tool of choice for simple submissions, especially when only one or a small number of records is submitted (9).

BankIt can also be used by submitters to update their existing GenBank records.


NCBI has developed a stand-alone multi-platform submission program called Sequin (http://www.ncbi.nlm.nih.gov/Sequin/ index.html ) which can also be linked online to NCBI.

Sequin handles simple sequences (e.g. a cDNA), as well as long sequences and segmented entries, for which BankIt and other Web-based submission tools are not well-suited.

Sequin has convenient editing and complex annotation capabilities and contains a number of built-in validation functions for enhanced quality assurance.


It is also designed to facilitate the submission of sequences from phylogenetic, population and mutation studies, and can incorporate alignment data.

Sequin can be used to edit and update sequence records, as well as to perform sequence analysis.

For example, Sequin can now incorporate any analysis tool available on the Web that accepts FASTA or ASN.1 (Abstract Syntax Notation 1) formatted data as its input.


In addition, Sequin is able to work on large records (e.g., the Escherichia coli genome at 5.6 Mb) and read in all of its annotations via simple tables.

Versions for Macintosh, PC and Unix computers are available via anonymous FTP to 'ncbi.nlm.nih.gov' in the 'sequin' directory.

Once a submission is completed, users can Email it to the address: gb-sub@ncbi.nlm.nih.gov .

Additional information about Sequin can be found through the NCBI home page.


Entrez (http://www.ncbi.nlm.nih.gov/entrez/ ) is an integrated database retrieval system that accesses DNA and protein sequence data, genome mapping data, population sets, the NCBI taxonomy, protein structures from the Molecular Modeling Database, MMDB (11) and MEDLINE references via PubMed.

The DNA and protein sequence data are integrated from a Nucleic Acids Research, 2000, Vol. 28, No. 1 variety of sources and therefore include more sequence data than are available within GenBank alone.

Entrez searching is provided on NCBI's Web site, via the Query Email server (query@ncbi.nlm.nih.gov ), and as a network client that can be downloaded by FTP.

Entrez is also discussed elsewhere in this issue (4).


The most frequent type of analysis performed using GenBank is the search for sequences similar to a query sequence.

NCBI offers the BLAST (http://www.ncbi.nlm.nih.gov/BLAST/ ) family of programs to locate good alignments between a query sequence and database sequences (12,13).

BLAST searching is provided on NCBI's Web site, via an Email server (blast@ncbi.nlm.nih.gov ), and as a set of stand-alone programs distributed by FTP.

BLAST is discussed in more detail in a separate article in this issue (4).


NCBI uses the ASN.1 data format for internal maintenance of GenBank, but distributes the GenBank releases in the traditional flat-file format.

The full GenBank release (issued every 2 months) and the daily updates (which also incorporate sequence data from EMBL and DDBJ) are available by anonymous FTP from 'ncbi.nlm.nih.gov'.

The full release in flat-file format is available as compressed files in the directory, 'genbank'.

A cumulative update file is contained in the sub-directory, 'daily', and a non- cumulative set of updates is contained in 'daily-nc'.

A set of sequence-only files in FASTA format, corresponding to the GenBank database subsets searched by BLAST and including the non-redundant nucleotide and protein databases, is available in the 'blast/db' directory.

Software developers creating their own interfaces or analysis tools for GenBank data are offered the NCBI ToolKit to assist in developing specialized applications.

The ToolKit software can be found in the directory 'toolbox/ ncbi_tools'.


In addition to maintaining the GenBank nucleic acid sequence database, the National Center for Biotechnology Information (NCBI) provides data analysis and retrieval resources that operate on the data in GenBank and a variety of other biological data made available through NCBI’s web site.

NCBI data retrieval resources include Entrez, PubMed, LocusLink and the Taxonomy Browser.

Data analysis resources include BLAST, Electronic PCR, OrfFinder, RefSeq, UniGene, HomoloGene, Database of Single Nucleotide Polymorphisms (dbSNP), Human Genome Sequencing, Human MapViewer, Human-Mouse Homology Map, Cancer Chromosome Aberration Project (CCAP), Entrez Genomes, Clusters of Orthologous Groups (COGs) database, Retroviral Genotyping Tools, SAGEmap, Gene Expression Omnibus (GEO), Online Mendelian Inheritance in Man (OMIM), the Molecular Modeling Database (MMDB) and the Conserved Domain Database (CDD).

Augmenting many of the web applications are custom implementations of the BLAST program optimized to search specialized data sets.

All of the resources can be accessed through the NCBI home page at http://www.ncbi.nlm.nih.gov.


The National Center for Biotechnology Information (NCBI) at the National Institutes of Health was created in 1988 to develop information systems for molecular biology.

In addition to maintaining the GenBank [1] nucleic acid sequence database, to which data is submitted directly by the scientific community, NCBI provides data retrieval systems and computational resources for the analysis of GenBank data and the variety of other biological data made available through NCBI.

For this update, the NCBI suite of database resources is grouped into the seven categories given below.

All resources discussed below are available from the NCBI home page at http://www.ncbi.nlm.nih.gov.


Entrez [2] is an integrated database retrieval system that accesses DNA and protein sequences derived from several sources [1,3–6], genome maps, population sets, protein structures from the Molecular Modeling Database (MMDB) [7] and the biomedical literature via PubMed and Online Mendelian Inheritance in Man (OMIM), with embedded links to the NCBI taxonomy.

PubMed includes primarily the 11 million references and abstracts in MEDLINE, with links to the full-text of more than 1100 journals available on the web.

Several new features have been added to Entrez recently including searches for proteins by molecular weight range or by protein domain.

Population sets and ‘ProbeSet’ data from gene-expression experiments are also now accessible from Entrez.

New Entrez formatting options include graphical and text-mode display of PHRAP scores for HTG records and XML output for all GenBank records.

The Books database has been integrated into Entrez allowing the searching of three online scientific textbooks [8–10].


The LocusLink database of official gene names and other gene identifiers [6], was developed at NCBI in conjunction with several international collaborators, and offers a single query interface to curated sequences and descriptive information about genes.

New links from the LocusLink report lead to Proteome Inc.’s Gene Ontology, and NCBI’s Evidence Viewer which shows the alignments between GenBank sequences and the NCBI Human Genome Assembly used to derive alignment-based gene models.

Protein domains from NCBI’s Conserved Domain Database (CDD) that are detected in gene translation products are also now listed in the LocusLink report.


The Basic Local Alignment Search Tool (BLAST) family of programs [11,12] performs sequence-similarity searches, beginning with either a query sequence or a GenBank accession number.

Successful searches return a set of gapped alignments between the query and similar database sequences, with links to the full database records.

Each alignment receives a score and a measure of statistical significance, called the Expectation Value, for judging its quality.

The NCBI BLAST interface has been re-designed and offers several new search options including the specification of an Expectation Value range, rather than a threshold, for reporting alignments, and the specification of a residue range to limit searches to a portion of the query sequence.

XML output is now supported.

A new alignment format, called the ‘Hit Table’, provides a compact, tabular summary of the BLAST results including, for each database hit, the positions of alignment starts and stops, coupled with scores and Expectation Values.

In addition, BLAST can generate a taxonomically organized output that shows the distribution of BLAST hits by organism in three formats.

A particularly powerful feature of the new BLAST interface allows searches to be restricted to a database subset using standard Entrez search strings; the same restrictions may be applied to screen the output of an initially unrestricted search.

These features provide the means to effectively construct a custom database for searching, or to parse the output of a search to include only sequences of interest, respectively.

Along with the revised BLAST interface, NCBI has implemented a standard URL-API which allows complete search specifications, including BLAST parameters and search query, to be contained in the URL posted to the web page.

A ‘GetURL’ button on the BLAST pages allows for the saving of the current parameter set, but URLs for custom searches may also be constructed easily by users.

On the algorithmic side, BLAST now takes into account the amino acid composition of the query sequence in its estimation of statistical significance.

A composition-based statistical treatment, used in conventional protein BLAST searches as well as PSI-BLAST [12] searches, tends to reduce the number of false-positive database hits [13].

A useful BLAST utility, BLAST2Sequences [14], compares two DNA or protein sequences and produces a dot-plot representation of the alignments it reports.

Translated search options, such as blastx, tblastx and tblastn, now extend the program’s range beyond blastn and blastp.

Using a new nucleotide BLAST variant, called MegaBLAST [15], batches of nucleotide sequences can be pasted into a web page or uploaded from a file, and used to search for nearly exact matches in nucleotide databases.

MegaBLAST is up to 10 times faster than BLASTn for such searches.


NCBI has developed a semi-automated system for assembling both finished and unfinished human genome sequence on a regular basis so as to incorporate the most current data.

The NCBI-generated assembly of the human genome may be searched via a specialized variant called Human Genome BLAST using either nucleotide or protein queries.

Human Genome BLAST generates custom ‘Genome view’ of the BLAST hits which is integrated with the Human Genome MapViewer so that the hits can be viewed in the context of a combination of the maps used by the MapViewer, such as maps showing confirmed and predicted gene locations, or EST hits.

Finally, a special database, called the Trace Archive, which contains raw data underlying sequences generated by the various genome projects, may be searched using MegaBLAST.

The Trace Archive contains Whole Genome Shotgun (WGS) reads from the mouse as well as data from rat, human, zebrafish and worm.


BLAST-Link (Blink) is a new resource that displays pre-computed protein BLAST alignments for each protein sequence in the Entrez databases.

Blink allows for the display of subsets of these alignments by taxonomic criteria, by database of origin, relation to a complete genome, membership in a COG [16] or by relation to a 3D structure or conserved protein domain.

Blink links are displayed for protein records in Entrez as well as within LocusLink reports.


UniGene [17] is a system for automatically partitioning GenBank mRNA sequences and EST, into a non-redundant set of gene-oriented clusters with identical 3′ untranslated regions (3′ UTRs).

Six new organisms have been added to UniGene over the last year: barley, rice, Thale cress, maize, frog and wheat.

These organisms join human, mouse, rat, zebrafish and cow, bringing the total number of organisms represented to 11.

The UniGene databases are updated weekly with new EST sequences, and bimonthly with newly characterized sequences and may be downloaded by FTP.


HomoloGene is a database of both curated and calculated gene orthologs and homologs for the human, mouse, rat, zebrafish, cow, frog and fly; the latter two organisms added over the last year.

There are now over 50,000 homology groups for human and mouse.

The current datasets for the calculated orthologs and homologs and Mutually Orthologous Pairs are available via FTP.


The References Sequence (RefSeq) database [6], provides curated reference sequences for mRNAs and proteins from human and other organisms.

A new type of RefSeq, with accession number of the form NG_######, has been created to accommodate curated genomic regions.

Such regions now include the human hair keratin gene cluster on chromosome 17, the human α globin region on chromosome 16, and the human MHC class III complement gene cluster on chromosome 6, among others.


The database of Single Nucleotide Polymorphisms (dbSNP) [18] serves as a repository for both single base nucleotide substitutions and short deletion and insertion polymorphisms and now contains almost 3 million human SNPs.

A new web interface allows flexible searches by gene name and by cross-reference to other databases such as OMIM or the structure databases.

Searches for SNPs lying between two markers and batch retrieval are now also supported.


Electronic PCR (e-PCR) is a tool for locating STSs within a nucleotide sequence by comparing the query against a database of STS sequences and primer pairs.

A new non-redundant database of over 133,000 human and 80,000 non-human STSs called UniSTS is now available for such comparisons.


The Human Genome MapViewer displays NCBI’s current human genome assembly using up to seven parallel chromosomal maps simultaneously.

The maps displayed can be selected from a set of 21, and include cytogenetic maps, such as chromosomal ideograms, sequence-based maps, such as those showing contigs, genes and SNPs, and radiation hybrid maps, such as the G3 and GB4 maps.

Several new maps have recently been added to show ab initio gene models predicted by GenomeScan, EST alignments with links to UniGene clusters and mRNA alignments used to construct gene models.

The MapViewer also offers a tabular view of the data, in addition to the default graphical view, which is convenient for the import of the data into other programs for further analysis, and can be used to download user-selected segments of the NCBI Human Genome Assembly using a ‘Download/View Sequence’ link.

Supported download formats are GenBank and FASTA.

The NCBI Human Genome Assembly data are also available via FTP.

The Human Genome Resources web page collects links to all of NCBI’s human genome-related tools on one page.

A recent addition to these links is the Human BAC Resource, a catalog of large-insert, FISH-mapped clones containing sequence-tagged sites that can be used to integrate cytogenetic, radiation-hybrid, linkage and sequence maps of the human genome and to locate clone distributors.


The Cancer Chromosome Aberration Project (CCAP) service is an initiative of the National Cancer Institute (NCI) and NCBI.

The data includes a compilation by F. Mitelman, F. Mertens and B. Johansson of recurrent neoplasia-associated chromosomal aberrations from the Cancer Chromosome Aberration Bank at the University of Lund, Sweden [19].

A new Spectral Karyotyping database, SKY, has been created jointly by NCI and NCBI to enable investigators to share their own SKY and Comparative Genomic Hybridization (CGH) data on chromosomal aberrations (http://www.ncbi.nlm.nih.gov/sky/skyweb.cgi).


Entrez Genomes [20] provides access to genomic data contributed by the scientific community for over 900 species whose sequencing and mapping is complete or in progress, and now includes more than 57 complete microbial genomes and 245 reference sequences for eukaryotic organelles; a gain of about 27 microbial genomes and 80 organelle sequences over last year.

Four higher eukaryotic genomes are also found within Entrez Genomes; Homo sapiens, Drosophila melanogaster, Caenorhabditis elegans and Arabidopsis thaliana.

A new tool for genomic comparisons has been added within the last year called TaxPlot, which graphically compares similarities in the proteomes of two organisms to that of a third reference organism.

Entrez Genomes reports for complete microbial genomes give access to views showing various aspects of proteome organization, including the taxonomic distribution of proteins encoded in the genome (TaxMaps) and their membership in Clusters of Orthologous Groups (COGs) [16], links to 3D structures and conserved protein domains.

Pairwise sequence alignments for sequence neighbors are presented graphically and linked to the Cn3D macromolecular viewer [21], which allows the interactive display of 3D structures and sequence alignments.


The COGs database [16] presents a compilation of orthologous groups of proteins from completely sequenced organisms representing 30 phylogenetically distant clades.

Many new organisms have been added over the last year, bringing the total to over 44.

The COGs are now also linked to the proteins of two higher eukaryotes; the worm and the fly.


NCBI’s SAGEmap service implements many functions useful in the analysis of SAGE data such as a two-way mapping between SAGE tag and UniGene.

A new Java-Based SAGEmap Submission Tool (SST) is now available to assist SAGEmap submissions, and may be useful as a SAGE data organizational and analysis tool.


NCBI provides web access to the OMIM catalog of human genes and genetic disorders authored and edited by Victor A. McKusick at The Johns Hopkins University [22].

The database contains information on disease phenotypes and genes, including extensive descriptions, gene names, inheritance patterns, map locations and gene polymorphisms.

OMIM currently contains 12,940 entries, including data on 9,526 established gene loci and 926 phenotypic descriptions, and is now searchable using the powerful Entrez interface.


The Gene Expression Omnibus (GEO), a data repository and retrieval system for gene expression data, is described in a separate article in this issue [23].


The NCBI MMDB, built by processing entries from the Protein Data Bank [5], is described by Wang et al. [ 7].

The structures in the MMDB are linked to sequences in Entrez and to the CDD.

The CDD contains PSI-BLAST-derived Position Specific Score Matrices (PSSM’s) representing domains derived principally from two public protein domain collections, the Simple Modular Architecture Research Tool (SMART) [24] and Pfam [25], but also draws from domains defined by NCBI researchers.

NCBI’s Conserved Domain Search (CD-Search) service can be used to search a protein sequence for conserved domains in the CDD.

Wherever possible CDD hits are linked to structures which, coupled with a multiple sequence alignment of representatives of the domain hit, can be viewed with NCBI’s 3D molecular structure viewer, Cn3D [21].

A new Tool, called the Domain Architecture Retrieval Tool (DART), has been added to the MMDB suite which allows searches of protein databases on the basis of a combination of conserved domains.

Protein domain information has also been integrated into the Entrez system as mentioned above.


Most of the resources described here include documentation, other explanatory material and references to collaborators and data sources on the respective web sites.

Several tutorials are also offered under the Education link from NCBI’s home page.

A Site Map provides a comprehensive table of NCBI resources, and the What’s New feature announces new and enhanced resources.

A user support staff is available to answer questions at info@ncbi.nlm.nih.gov.


The GenBank sequence database incorporates publicly available DNA sequences of more than 105,000 different organisms, primarily through direct submission of sequence data from individual laboratories and large-scale sequencing projects.

Most submissions are made using the BankIt (web) or Sequin programs and accession numbers are assigned by GenBank staff upon receipt.

Data exchange with the EMBL Data Library and the DNA Data Bank of Japan helps ensure comprehensive worldwide coverage.

GenBank data is accessible through NCBI’s integrated retrieval system, Entrez, which integrates data from the major DNA and protein sequence databases along with taxonomy, genome, mapping, protein structure and domain information, and the biomedical literature via PubMed.

Sequence similarity searching is provided by the BLAST family of programs.

Complete bimonthly releases and daily updates of the GenBank database are available by FTP.

NCBI also offers a wide range of World Wide Web retrieval and analysis services based on GenBank data.

The GenBank database and related resources are freely accessible via the NCBI home page at http://www.ncbi.nlm.nih.gov.


GenBank is a public database of all known nucleotide and protein sequences with supporting bibliographic and biological annotation, built and distributed by the National Center for Biotechnology Information (NCBI), a division of the National Library of Medicine (NLM), located on the campus of the US National Institutes of Health (NIH) in Bethesda, MD [1].

NCBI builds GenBank primarily from the direct submission of sequence data from authors.

Another major source of data is bulk submission of EST, GSS and other high-throughput data from sequencing centers.

The US Office of Patents and Trademarks (USPTO) also contributes sequence data from issued patents.

The data are supplemented by sequences submitted to other public databases.

Through a long-standing international collaboration with the EMBL Data Library [2] in the UK and the DNA Databank of Japan (DDBJ) [3], data are exchanged daily to ensure that all three sites maintain a comprehensive collection of sequence information.

NCBI makes the data available at no cost over the Internet, by FTP access and by web text and sequence similarity search services.

NCBI also offers a wide range of World Wide Web retrieval and analysis services which operate on the GenBank data [4].


Obstetric anal sphincter injuries (OASIS) are serious complications to vaginal deliveries, with a 50% long-term risk of anal incontinence, that is, involuntary loss of feces or flatus.

1-3 In Norway, a National preven- tion program aiming at reducing the incidence of OASIS was initiated in 2004.

The prevention program was inspired by the management of deliveries in Finland, where the incidence of OASIS at that time was very low.

4 The program consisted of manual protection of the peri- neum, visualization of the perineum during crowning of the fetal head, focus on good communication with the delivering woman and use of episiotomy only on indication (the Norwegian/Finnish model).

5 After a couple of years, the incidence of OASIS in Norway dropped signifi- cantly, from 4%-5% to 1%-2%.

5 In Denmark, the incidence of OASIS in primiparous women in- creased from 6.1% in 2000 to 7.4% in 2010.

6 Unlike the situation in Norway, there has been no decision on a national OASIS-prevention program in Denmark.

However, since 2012, the incidence of OASIS has been monitored and evaluated yearly in a national setting (Danish National Quality Database for Births).

7 Despite no national strategy for OASIS prevention, the overall incidence of OASIS among first-time mothers has decreased in Denmark since the detailed national surveil- lance started in 2012; from 6.2% in 2012 8 to 4.2% in 2017.

7 In 2015, the Danish Society of Obstetrics and Gynecology pub- lished a national guideline 9 on evidence to prevent and thereby reduce the incidence of OASIS.

The recommendation was that all obstetric departments in Denmark should initiate OASIS-preventive strategies.

Before publication of the guideline, two of four obstetric departments in the Capital Region of Denmark had already initiated formal prevention programs.


The prevention programs were inspired by the Norwegian/Finnish model, which is further described in the Material and methods section.


The aim of the present study was to evaluate the effect of pre- ventive initiatives to reduce the incidence of OASIS by comparing hospitals from the same region with and without formal prevention programs and by comparing risk factors over time and at the differ- ent departments.


We performed a historical cohort study, evaluating incidence, change of incidence and risk factors of OASIS during the years 2011-2015 at the four delivery departments in the Capital Region of Denmark.

Two of the departments at Herlev (Hospital 1) and Hillerød (Hospital 2) Hospitals used formal prevention programs to reduce the risk of OASIS, adopting the Norwegian/Finnish model, but Hvidovre Hospital (Hospital 3) and Rigshospitalet (Hospital 4) did not introduce a formal prevention program during the study period.

We used data from the Danish National Birth Registry.


The main outcome of this study was the incidence of OASIS.

Variables included in the analyses to test for association with the in- cidence of OASIS were: year of delivery (calendar year [2011-2015], years after and before intervention [2014 and 2015 vs 2011 and 2012, deliveries in 2013 were excluded in the analyses of the effect of formal prevention programs as education and certification took place during 2013]), hospital (separately and grouped by use of for- mal prevention program [yes/no] [Hospitals 1 and 2 vs Hospitals 3 and 4], maternal age at delivery [years]), maternal prepregnant body mass index (kg/m 2 ) (as continuous variable and categorized as under- weight [<18.5], normal weight [18.5-24.9], overweight [25-29.9] and obese [>30]), maternal smoking (yes/no), primiparity, fetal birthweight (g or kg), fetal head circumference (cm), gestational age (days), fetal presentation (occiput anterior, occiput posterior and breech delivery), 5-minute Apgar score <7, induction of labor, oxytocin stimulation, epidural anesthesia, vacuum extraction, forceps delivery, mediolateral episiotomy, and time of delivery (daytime [08.00-23.59 hours] or night [00.00-07.59 hours]).


At Herlev Hospital (Hospital 1), the formal prevention program started with several meetings for midwives and doctors at the de- livery ward to inform about the diagnosis and prevention of OASIS.

A Norwegian midwife who had worked with the Norwegian pre- vention program, 10 participated for 2 weeks in the clinical work and instructed colleagues at the department, focusing on bedside learn- ing and "hands-on".

Twelve midwives were trained and certified in the Norwegian/Finnish techniques.

Subsequently, these 12 midwives trained the rest of the department's midwives, obstetricians, gynecol- ogists and residents.

Furthermore, the obstetrician who implemented the preventive model in Norway, 4 held a "hands-on" training session where the manual protection technique in vacuum-assisted deliveries was practiced on a phantom.

Each midwife and doctor, regardless of previous training skills and experience, was supervised at a minimum of three deliveries before certification.

After implementation of the formal prevention program was completed, all newly hired midwives and doctors, irrespective of previous training, were obliged to take part in an education session with phantom training, as well as super- vised deliveries before certification.


At Hillerød Hospital (Hospital 2), the prevention program was initiated in 2010 and included courses in correct handling of OASIS (theory, diagnosis and suturing).

From May 2012, hands-on tech- nique courses were held for midwives and obstetricians.

The protec- tive technique was optional and could be according to the "Finnish" technique, 11 Ritgens maneuver 12 or another manual protection technique.

As this prevention program did not seem to reduce the number of OASIS significantly (data not shown), all staff attended a 1-day course introducing the technique used in the Norwegian


During the years with a general focus on how to prevent obstetric anal sphincter injuries, the incidence decreased significantly.

Hospitals with formal prevention programs, however, did not have a higher reduction in incidence than hospitals without programs.

16000412, 2019, 11, Downloaded from https://obgyn.onlinelibrary.wiley.com/doi/10.1111/aogs.13672 by Mcgill University, Wiley Online Library on [18/05/2024].

See the Terms and Conditions (https://onlinelibrary.wiley.com/terms-and-conditions) on Wiley Online Library for rules of use; OA articles are governed by the applicable Creative Commons License JANGÖ et Al.

model performing hands-on training on phantoms.

Training was con- ducted by both a Finnish midwife and obstetrician who participated in the Norwegian national preventive program.

Back at the delivery ward, all midwives and obstetricians (irrespective of previous train- ing, skills and experience) were supervised by certified local staff.

Supervision of the midwives included a minimum of three training sessions on a phantom followed by supervision on three deliveries before certification.

The doctors were also supervised in "hands-on" vacuum extraction on a phantom, using the Norwegian model, re- quiring three supervised sessions on a phantom before certification.


Hvidovre Hospital (Hospital 3) and Rigshospitalet (Hospital 4) served as controls, as these hospitals did not have a formal OASIS- prevention program in the time period.

At Hospital 3, OASIS-pre- ventive initiatives started during 2015, focusing on diagnosis, episiotomy technique and voluntary training sessions.

No certifica- tion or bedside training was applied.

Hospital 4 did not have a formal prevention program for OASIS.


Statistical analyses to evaluate changes in incidence of OASIS and risk factors over time were made using trend tests.

To compare groups, we used the Chi-square test, Fisher's exact test and Kruskal- Wallis test as appropriate.

Further, we evaluated risk factors of OASIS using uni- and multivariable analyses.

The multivariable logis- tic regression analyses were performed as stepwise regression with backward elimination of nonsignificant or borderline nonsignificant variables.


Since the incidence of OASIS has previously been found to be influenced by whether episiotomy is performed in deliveries with and without vacuum extraction, 6 we tested for interaction be- tween these factors.

We also tested for interaction between year of delivery (2014 and 2015 vs 2011 and 2012) and for hospital with or without formal prevention programs.

Significance level was set as a P-value <0.01 due to the large number of analyses and therefore risk of multiplicity.


In the final multivariable model, the analyses were adjusted for the following variables: year of delivery, place of birth at department with or without formal prevention program, mater- nal age, smoking during pregnancy, primiparity, fetal birthweight, epidural anesthesia, vacuum extraction and episiotomy.

Forceps, although significantly associated with the risk of OASIS, were not included in the multivariable analyses since forceps are rarely used in Danish obstetric practice.

Statistical analyses were performed using the statistical software R (https ://www.r-proje ct.org/).


We used registry data from the Danish Medical Birth Registry (Permission No. FSEID-00002666, date of approval 19 January 2017).

The study was also approved by the National Data Protection Agency (J.nr.

HGH-2016-035, I-Suite nr. 04489.

Date of approval: 1 March 2016).


There were 93 273 deliveries in the study period.

Of these, 75 173 (80.6%) were vaginal deliveries and 18 100 (19.4%) were by cesarean section.

Among vaginal deliveries, 2670 (3.6%) were complicated by OASIS.

The incidence of OASIS decreased during the study period from 4.3% (n = 636) in 2011 to 2.6% (n = 399) in 2015 (Table 1).


A significant decrease was found for all four hospitals during the study period (Figure 1).

However, comparing the annual incidence of OASIS between the four hospitals, we found that the incidence was equal in years 2011 and 2012, but that there was a significant dif- ference between the hospitals during the years 2013-2015 (Table 1).


During the study period, the cesarean section rate did not change significantly (P = 0.79), nor did the rate of vacuum-assisted deliveries (P = 0.05).

However, we found a significant change in the proportion of primiparous women over the study period.

At the hospitals with formal prevention programs, the proportion of primiparous women increased from 36.6% to 43.1% (P < 0.001) and at hospitals without formal pre- vention programs, the proportion decreased from 57.6% to 54.9% (P < 0.001).


There was a significant increase in the rate of episiotomy in the study period from 3.8% in 2011 to 5.5% in 2015 (P < 0.001) and this increase was significant both at hospitals with formal pre- vention programs (P < 0.001) and those without (P = 0.001).

Further, the episiotomy rate differed at hospitals with and without formal pre- vention programs.

Notably, the rate of episiotomy in vacuum-assisted deliveries increased markedly at hospitals with formal prevention pro- grams, although a similar but smaller increase was found in hospitals without formal prevention programs (Figure 2).


When comparing the hospitals in the years after intervention (2014 and 2015), we found a lower risk of OASIS in spontaneous deliveries without episiotomy at hospitals with formal prevention program (1.5%, n = 158) than at hos- pitals without formal prevention programs (2.0%, n = 331; P = 0.002) (Table 2).

In vacuum-assisted deliveries at hospitals with formal pre- vention programs, the rate of episiotomy increased from 9.8% in 2011 to 27.9% in 2015.

After implementation of the formal prevention pro- grams, the risk of OASIS was 7.7% if episiotomy was performed in vac- uum-assisted deliveries and 10.0% without episiotomy, although this difference was not significant (P = 0.27).

At hospitals without formal prevention programs, the rate of episiotomy in vacuum-assisted deliv- eries increased from 8.9% in 2011 to 17.5% in 2015.

At the time after intervention at the other hospitals, the hospitals without formal pre- vention programs had the same risk of OASIS irrespective of whether episiotomy was used (10.7% vs 10.7%, P = 0.99).


In Table 3, population characteristics according to hospital with and without formal prevention programs at the years after implementation (2014-2015) are presented.

Notably, there was a significantly higher proportion of primiparous women at the two de- partments without formal prevention programs (55.8%) than at the departments with such programs (39.5%; P < 0.001).

We also found F I G U R E 1 Changes in incidence of obstetric anal sphincter injury (OASIS) over time at the four Hospitals in the Capital Region 2011-2015 [Color figure can be viewed at wileyonlinelibrary.com] When analyzing risk factors of OASIS during the study period we found two important interactions.

First, we found that year of delivery (2014 and 2015 vs 2011 and 2012) and whether the de- partment had a formal prevention program affected the risk of OASIS differently (P = 0.008).

Secondly, we found that the risk of OASIS was affected differently in vacuum-assisted deliveries with or without episiotomy (P < 0.001).

Therefore, these two interactions were included in both the univariable and the multivariable analyses (Table 4).

After adjustment and removal of nonsignificant and bor- derline significant/nonsignificant variables (close to P = 0.01), both interactions remained significant.


In the univariable analyses, we found that the risk of OASIS during 2011 and 2012 did not differ significantly between hospitals with and without formal prevention programs (P = 0.15).

When com- paring the risk of OASIS at departments without formal prevention programs in 2011 and 2012, there was a significantly lower risk in 2014 and 2015 for hospitals without formal prevention programs (crude odds ratio 0.67, 95% confidence interval [CI] 0.60-0.75) and an even lower risk at hospitals with formal prevention programs (crude odds ratio 0.48, 95% CI 0.42-0.55).


The final multivariable analyses were adjusted for year of de- livery, formal prevention program, maternal age, smoking status, parity, fetal birthweight, induction of labor, epidural anesthesia, vac- uum extraction and episiotomy.

After multivariable adjustments we found a higher risk of OASIS at the departments with formal preven- tion programs at baseline (years 2011 and 2012) (adjusted odds ratio [aOR] 1.36, 95% CI 1.20-1.54) than departments without formal prevention programs.

Similar to the univariable analyses, we found a reduced risk of OASIS in 2014 and 2015 for both the departments with formal prevention programs (aOR 0.67, 95% CI 0.58-0.78) and those without (aOR 0.68, 95% CI 0.61-0.76).


The risk of OASIS in vacuum-assisted deliveries was higher if performed without episiotomy (aOR 3.08, 95% CI 2.77-3.43) than if it was performed with episiotomy (aOR 2.35, 95% CI 1.89-2.93).

In the multivariable analyses we also found that increasing mater- nal age, primiparity, increasing birthweight and epidural anesthesia increased the risk of OASIS.

Smoking during pregnancy significantly decreased the risk of OASIS, even after adjustment for the other variables, including fetal birthweight.


We performed a historical cohort study with prospective follow up to evaluate the change in incidence of obstetric anal sphincter injury (OASIS) at four delivery departments in the Capital Region of Denmark and found that the overall incidence of OASIS decreased significantly during the period, at both the departments with and those without formal prevention programs.

The adjusted analyses showed a similar risk reduction at departments with and without formal prevention pro- grams when controlling for important risk factors and for calendar year.


Until recently, the Nordic countries and UK have had increas- ing incidences of OASIS.

6,13-15 One hypothesis for this could be a previous tendency towards "hands off" the perineum; 16,17 another could be the increased focus on diagnosing and coding of OASIS.

13 The convincing large-scale observational data with substantial re- duction in the incidence of OASIS in Norway due to a national strat- egy of OASIS prevention inspired several departments in Denmark to initiate local prevention programs.

18,19 In Norway, there was a significant decrease from 4%-5% to 1%-2% OASIS in all vaginal deliveries.

20,21 At one of the hospitals in Norway the incidence of OASIS decreased from 4% to 1% over a few years after adopting the Norwegian/Finnish model.

4 Similarly, at the Danish hospitals using the Norwegian/Finnish-inspired formal prevention programs, our study showed that the incidence dropped from around 4% to 2%.

However, even at the two hospitals without formal prevention pro- grams in this study, the incidence decreased, although there was a significant difference in the incidence of OASIS at the hospitals with and without formal prevention programs after the interventions.


The robustness of evidence regarding perineal protection and reduction of OASIS using the Norwegian model have previously been criticized.

22,23 The randomized trials performed to evaluate perineal protection have failed to provide effective reduction in the TA B L E 2 Incidence of obstetric anal sphincter injury (OASIS) in spontaneous and vacuum-assisted deliveries depending on episiotomy and formal prevention program in the years after intervention (2014-2015) (n = 30 835) OASIS rate.

24 The Norwegian/Finnish model includes both perineal protection and other preventive strategies, and therefore it is diffi- cult to differentiate between the specific strategies and how these affect the overall risk of OASIS, separately and combined.

22 Based on our results it seems that some cases of OASIS can be prevented when adopting general focus on prevention strategies.


However, we found no significant extra effect of implementation of a formal training program.

The decreased incidence of OASIS did not reach the magnitude reported from Norway.

In Denmark, prevention strategies depended on local initiatives.

In contrast, the marked reduction of OASIS in Norway was the result of a na- tional strategy with compulsory tutorials in all clinics.

In Norway, TA B L E 4 Risk factors of obstetric anal sphincter injury (OASIS) according to maternal, fetal and obstetric characteristics presented as uni- and multivariable analyses It is possible that this could have caused a more marked effect on obstetrical practice, resulting in a higher reduction of incidence of OASIS, causing a significant difference between the hospitals with and without formal prevention programs.


An intervention program like the Norwegian program is costly.

Further, the preventive effect is not verified by randomized controlled trials.

These may be expla- nations for not implementing intervention programs like the one used in Norway.

It is possible that future rigorous prevention strate- gies will significantly alter the incidence of OASIS.

The results from Norway with a continuously low number of OASIS is admirable, and taking the severe long-term complications to OASIS into consider- ation, continuous focus and prevention is needed to further reduce the number of women with OASIS.


One of the side-effects of the increased focus on reduction in the incidence of OASIS was the increased use of episiotomy.

In Denmark, the episiotomy rate has been decreasing 6 and Blondel et al found that Denmark had the lowest episiotomy rate (3.7%) of the European countries.

25 In the present study, we found that the episiotomy rate increased significantly after implementation of the formal prevention programs and to a lesser extent also at the hospitals without formal prevention programs.

Especially, the rate of episiotomy increased in vacuum-assisted deliveries, possi- bly as a result of recent data that this protects against OASIS in primiparous women.

26 In Norway, education in episiotomy tech- nique was one of the central parts in the prevention of OASIS.

Laine et al found that the rate of episiotomy actually decreased in spontaneous deliveries but increased significantly in instrumental deliveries in primiparous women (from 61% to 85%).

20 Although we found an increased risk of episiotomy rate in vacuum-assisted de- liveries, the episiotomy rate in our study is substantially lower than that in the Norwegian study.

The results of an ongoing Swedish randomized controlled trial that is evaluating the influence of lat- eral episiotomy in vacuum-assisted deliveries among primiparous women are awaited.

27 The important strength of this study is the large sample size over a relatively short time in obstetric settings that are very similar for other matters than preventive strategies for OASIS.

The large data- set allows for multivariable testing to control for important risk fac- tors and possible confounders.

Data were collected for all deliveries and, ideally, bias due to registration as well as recall bias should be eliminated.


However, there are some weaknesses that need to be addressed.

First, the intervention with formal prevention programs was initi- ated and conducted differently at the two hospitals with prevention programs.

The Capital Region of Denmark is geographically small, and many midwives and doctors change work places within the re- gion.

Therefore, it is possible that the exchange of staff between hospitals with and without formal prevention programs might affect the incidence of OASIS during the study period.

Also, the national guideline on prevention of OASIS and the overall focus on the pre- ventive strategies may have had a Hawthorne effect 28 on the staff in hospitals without formalized preventive strategies.

Furthermore, many obstetric and demographic factors that could not be evaluated in our study, could have led to the effect of the formal prevention programs being too small to detect a significant difference.


In this study, we compared the effect of a formal prevention program with general recommendations.

We did not investigate the different prevention strategies separately (ie, perineal protection, communication, reducing speed of crowning and individualized need for episiotomy) but focused on the effect of the awareness on com- binations of these factors.


Further, we evaluated the effect on incidence of OASIS in the short term (few years) after the intervention.

Maintaining the lower incidence of OASIS over time requires continued focus, training and possibly also recertification, in order to preserve necessary clinical skills.


Hopefully, future studies will reveal which preventive strate- gies, separately or combined, can lead to the largest reduction in incidence of OASIS.

Nonetheless, until those studies have been per- formed, we are obliged to prevent as many cases of OASIS as possi- ble to avoid long-term consequences for the women affected.


We found that focusing on prevention of OASIS is associated with a decrease in incidence, but implementation of formal prevention programs did not provide an additional effect.

Possibly, more rigor- ous interventions at the hospitals with formal prevention programs might have caused a significant difference.


Sir,


Congratulations on an important work confirming that it is indeed feasible to react to a rising rate of obstetric anal sphincter injuries (OASIS) and reduce it [1].

Furthermore, you discuss whether the formal program or focus was the most important factor in reducing the rate.

This assumed contradiction is a misconception.

Focus is essential, regardless of the strategy.

Instead, the decisive questions to be answered are what factors made staff members focus, and what they focused on.


In general, one way to help people focus on a task such as implementing a hands-on approach in childbirth is to establish a formal prevention program.

Focus may also be supported by more informal, person-centered or contextual factors such as preparedness for the intervention, competition between departments, meetings, discussions on the internet, and proximity to a department with a program.

Fear of penalties is another important factor in favor of a systems approach.


Some departments in Denmark actually did not experience a fall in the incidence of OASIS despite a perceived focus and the nationwide guideline [2].

Focus may be counteracted by, for example, resistance of influential leaders.

Unfortunately, you did not document process indicators.

It may well be that more or less the same elements for handling delivery were taken up to about the same degree in all departments.

This would of course explain similar results.

A more thorough description of the different factors making the staffs focus, what they focused on, and how often this was applied would have been valuable [3].


You call for studies revealing which elements may lead to the largest reduction in the incidence of OASIS.

In our work, we registered process indicators, and we found the lowest incidence of OASIS in the group of primiparae where a certified midwife used a hand on the baby's head.

The single most important element of the care bundle was using a hand on the head [4].


Thus, in your case, both formal prevention programs and more informal and person-centered factors seem to have brought about a focus on a safer handling of delivery in the short term.

To achieve and sustain a low risk of OASIS in the long term, a formalized systems approach is needed [5].


Sir, We thank Dr. Rasmussen for his interest [1] in our paper [2].

As pointed out in the article, we recognize that formal obstetric anal sphincter injury (OASIS)-preventive programs will also increase the focus on reduction of the incidence of OASIS.

Furthermore, the publication of the Norwegian quality improvement projects has been the inspiration of OASIS-preventive practice in many other countries.

The study by Rasmussen et al. evaluating the different OASIS-preventive factors is interesting, because they, despite implementation of “a package” with certification, were able to analyze the importance of these factors separately [3].

It seems likely that their “package” resembles the one used in Norway, including visible perineum during crowning, manual perineal protection, a hand on the fetal head to slow down the delivery, and focus on communication.

The same factors were included at the two departments with formal prevention programs in our study, and similar to their intervention, those two departments also included compulsory certification of staff members.

The formal preventive programs were initiated by local midwives and obstetricians/residents, and both programs were appreciated by the leaders of the department.


Unfortunately, we do not have information about which preventive strategies staff members focused on at the departments without formal prevention programs.

We did not report or collect process indicators that could have increased our understanding of the process—both at the departments with and at those without formal prevention programs.

However, prospective collection of individual OASIS-preventive factors at all deliveries at the four departments over several years, would have been a tremendous amount of work and probably not feasible.

When evaluating obstetric practice and skills, it remains challenging to evaluate the results from multivariable analyses.

Different risk factors, protective factors and obstetric methods are often associated.

Hence, it is difficult to transfer results from single parameters to other staff members and other obstetric units.

Nevertheless, the finding that controlling the speed of crowning by placing a hand on the fetal head was the single factor that was most important [3], seems logical and important.

Along with the other preventive strategies, continuous focus on reducing speed of crowning is probably essential to reduce the risk of OASIS.


However, the finding that certification of midwives was essential could not be verified in our study.

Certification is time-consuming and costly, and along with the lack of evidence of perineal protection from randomized controlled trials, these were the reasons for not implementing such programs at the two departments without formal prevention programs.

Nevertheless, both individual and general focus are important, but it is plausible that more rigorous preventive programs can cause a sustained lower incidence of OASIS than in departments without such programs [4].

The difficulties associated with a valid OASIS diagnosis at delivery, and the association between OASIS and subsequent anal incontinence should be addressed.

Actually, increased focus on prevention of anal incontinence after delivery is essential.

Clinical assessment and endoanal ultrasound at follow up may provide clinical feedback and help us to understand how OASIS-preventive strategies affect the risk of anal incontinence after delivery.


Based on the institutional standards at Loma Linda University in Loma Linda, California, our study met the criteria for IRB exemption.

We did not receive any financial support for the research, authorship, and publication of this study.


A 2020 directory of former ophthalmology and otolaryngology (ENT) residency programs was obtained from the American Osteo- pathic Colleges of Ophthalmology and Otolaryngology-Head and Neck Surgery (AOCOO-HNS).

This directory represented AOA ENT and ophthalmology programs that existed before the single accreditation process began in 2014.


In January 2020, a secured survey was sent via direct e-mail to PDs of former AOA ENT and ophthalmology programs that did not achieve ACGME accreditation (Appendix).

Follow-up emails were sent on May 12, 2020 and again on May 22, 2020.

The survey helped reveal challenges faced by these programs, many of which were in cities with less than 50,000 people.

This survey examined why accreditation was not pursued or obtained utilizing two questions, each with response options generated through personal communication with AOA ENT and ophthalmology PDs.

Another response option with a comment box was provided for both questions.


(1) If you did not pursue ACGME accreditation, why did your program not pursue ACGME accreditation? ( Choose one or more of the following: excessive cost, excessive paperwork, lack of faculty support, lack of hospital/administration support, ACGME clinical case volume requirements, ACGME research and academic vol- ume requirements, ACGME logistical/facility proximity re- quirements, ACGME surgical case volume requirements, we did not pursue ACGME accreditation, and other.)


(2) If you did apply for ACGME accreditation, why did your program not attain ACGME accreditation? ( Choose one or more of the following: excessive cost, excessive paperwork, lack of faculty support, lack of hospital/administration support, ACGME clinical case volume requirements, ACGME research and academic vol- ume requirements, ACGME logistical/facility proximity re- quirements, ACGME surgical case volume requirements, we did not apply for ACGME accreditation, and other.)


Out of the 16 programs that were issued surveys, 12 re- spondents completed the surveys: six ophthalmology and six ENT PDs.

Ten (83.3%) of the respondents did not pursue ACGME accreditation, and 2 (16.7%) of the respondents pursued ACGME accreditation but were unsuccessful in achieving accreditation.

The most common reason behind the inability of the programs to pursue or achieve accredi- tation was the lack of hospital/administrative support (7; 58.3%).

Other frequently selected reasons included excessive costs and lack of faculty support (5; 41.7%). " Other" was chosen by 50% of respondents, citing common reasons associated with the cost-benefit of accreditation.

ACGME research and academic requirements (4, 33.3%), logistical/facility requirements (3; 25%), excessive paper- work (3; 25%), and surgical case volume requirement (2; 16.7%) were also cited as reasons for not pursuing ACGME accreditation.


Given the relatively small sample size in this survey, data from both ophthalmology and ENT respondents' "Other" reasons in their nonpursuit for ACGME accreditation were aggregated.

The ophthalmology program respondents who chose "Other" cited limited office space and independent third-party consultant recommendation as their reasons not to pursue accreditation from a cost-benefit perspective.

The hospital administration made a decision that reflected the consultant's recommendation.

The responses of ENT pro- grams varied.

One program was part of a hospital system that merged all residency programs with a nearby university and consolidated all specialties in primary care.

Another program closed its ENT residency to expand other residency programs.

One ENT PD stated that the sponsoring institution did not want to commit the time and money to achieve ACGME accreditation; furthermore, the PD believed that the program could have successfully attained ACGME accreditation had the hospital supported the ENT residency.


When the AOA initially debated the SAS, a major concern was regarding the ability of rural programs to achieve ACGME accreditation.

Nearly 30% of the 132 AOA training programs in rural locations did not achieve ACGME accreditation, the majority of which were primary care programs but also included surgical subspecialty fields.

Typically, the lack of access to physician care in rural communities is due to rural hospitals having trouble recruiting physicians and remaining financially viable.

Our survey results also reflect hospitals' financial issues.

Re- spondents highlighted the lack of logistical and adminis- trative support as well as the ongoing costs to maintain AOA surgical specialty programs as significant reasons for not pursuing/receiving ACGME accreditation.

We have provided four recommendations to gain a greater under- standing of and address this rural workforce issue.


Based on our survey results, the most common issues faced by former AOA surgical subspecialty programs that failed to receive ACGME accreditation were related to finances, logistics, and administration rather than clinical and sur- gical volumes.

Lack of quality in programs, such as those with smaller surgical and clinical case volumes, may be logical reasons for closing former programs.

However, it seems that surgical and clinical case volumes was not a common issue for most former AOA ENT and ophthal- mology programs because 3 of the 12 programs cited sur- gical or clinical volume as part of their decision not to pursue ACGME accreditation.


Considering these results, we have recommendations that should interest PDs and Designated Institutional Of- ficials who faced similar issues in other medical spe- cialties, hospital Chief Medical Officers that supervise program transitions, residents interested in how transi- tions toward ACGME accreditation could impact their future medical education, and health policy experts examining physician access in high-need areas.

The first recommendation is to expand Teaching Health Center Graduate Medical Education (THCGME).

Currently, the THCGME model has successfully established GME in high-need areas with high primary care physician retention rates.

However, no such program exists for surgical pro- grams despite increasing needs for surgical subspecialties in the rural workforce.

THCGME funding has been priori- tized to primary care specialties considering workforce supply and demand as well as an increasing national emphasis on primary care.

THCGME, however, now in- cludes surgical fields such as Obstetrics and Gynecology (Ob-Gyn), and existing Ob-Gyn programs can be utilized as a model to develop ENT and ophthalmology programs.


The second recommendation is to study other former AOA surgical subspecialty programs.

Our findings were specific to ENT and ophthalmology programs.

In similar fashion, 50% of former AOA neurosurgery programs transitioned to ACGME accreditation.

However, 90% of urology AOA programs are now ACGME accredited [4].

Systematic in- quiry is necessary to determine whether other affected surgical fields faced similar barriers transitioning to single accreditation.

It may be beneficial to study former AOA urology programs to determine which characteristics of the urology programs contributed to their high transition rate to the ACGME.

It is important to note that among the 10 urology programs that transitioned successfully, four were in cities with a population of less than 50,000.


The third recommendation is to address the lack of insti- tutional commitment and high cost to maintaining sub- specialty programs in high-need areas.

To a certain extent, we propose reexamining specific ACGME requirements to accommodate former AOA residency programs to increase surgical specialty care access in high-need areas.

Re- quirements surrounding academic output (research pro- jects and publications), faculty size, and distance between training sites may be reexamined to reduce costs and in- crease support for these programs.

To help facilitate this recommendation, we call upon the AOA and AACOM to address the previously mentioned ACGME requirements at the federal and ACGME levels.


The fourth recommendation is for the Centers for Medicare & Medicaid Services (CMS) to reconsider its physician reimbursement approach.

Currently, CMS periodically en- acts alterations to their fee schedule that involves the reallocation of funds between fields.

In the last several years, including the proposed fee schedule for 2021, CMS has enacted and proposed cuts to reimbursement for all surgical subspecialties [19].

Although increasing support from primary care specialties is essential and critical to public health, it should not come at the expense of surgical specialties.

Such decline in reimbursements also contrib- uted to ENT and ophthalmology program closures.

In our discussions with PDs, they often mentioned that the cost of enhancing programs to meet ACGME requirements was too high for institutions to justify the investment, especially when considering declining reimbursement from public insurance entities such as CMS.

Multiple institutions determined that it was more financially feasible to pursue their primary care program transitions.


Due to the small number of osteopathic ophthalmology and ENT PDs, there is a statistical limitation on the power of our study.

Other limitations include the lack of participa- tion of administrative stakeholders, the lack of more qualitative responses, and the lack of quantifiable data reflective of each respondent's hospital, community size, number of hospital beds, and department size.

We invited the PDs to participate in this study and did not extend the survey to hospital administrative leaders; hence, we only had the PDs' impressions to trust.

In our survey, we reserved a qualitative response section for PDs to express other thoughts and discussion, allowing further explana- tions regarding the reasoning behind their program's pursuit or nonpursuit for accreditation beyond the reasons they checked on the survey (e.g., costs, administrative support, research, surgical volumes, and other ACGME requirements).

However, we received limited responses in this category.


None reported.


All authors provided substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data; all authors drafted the article or revised it critically for important intellectual content; all authors gave final approval of the version of the article to be published; and all authors agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.


To the Editor:


We read Ahmed et al. ’s recent article “AOA Ophthalmology and Otolaryngology Program Closures as a Model to Highlight Challenges of Maintaining GME in High Need Areas” [1] with great interest and congratulate them on their publication.

They found that previously accredited AOA ophthalmology and otolaryngology residency programs were less likely to obtain ACGME accreditation (47 and 62%, respectively) than other previously accredited AOA surgical subspecialty residency programs, such as urology (90%) [1].


To contribute to their findings and respond to “Recommendation #2,” [1] we found that all 10 of the current urology residencies that successfully merged to ACGME are considered community-based or community-based/university-affiliated programs [2, 3].

Additionally, another study found that urology residents are more profitable than residents of other specialties [4].

This profitability could play a role in the decision to obtain accreditation for urology residency programs over ophthalmology or otolaryngology residency programs.


Two of the successfully transitioned urology residency programs are affiliated with a large university that was also affiliated with five AOA ophthalmology residency programs that never obtained accreditation after the ACGME merger [3, 5].

These five AOA ophthalmology residency programs contributed to 33% of all 15 AOA ophthalmology residencies in 2013 [5].

This accounts for more than half (62.5%) of the AOA ophthalmology residency programs that eventually did not obtain ACGME accreditation.

Because these five programs were all affiliated with one university, we believe that the AOA ophthalmology residency data presented by Ahmed et al. [ 1] may be misleading.

If the university had only been affiliated with one AOA ophthalmology residency program that never obtained accreditation, then the data would reflect that 7 out of 11 AOA ophthalmology residency programs would have received accreditation (64%), which is similar to that of AOA otolaryngology residency programs that successfully merged (62%) [1].


Despite these findings, we agree with the recommendations presented by Ahmed et al. [ 1] The ACGME merger appears to be a setback for osteopathic medical students interested in competitive specialties such as ophthalmology or otolaryngology.

We suggest further research that investigates potential barriers and incentives within a university that may cause it to choose certain surgical subspecialty residency programs (i.e., urology) over others (i.e., ophthalmology and otolaryngology).


To the Editor,


We appreciate the response from the authors [1] regarding our publication [2] on former American Osteopathic Association (AOA)-accredited ophthalmology and otolaryngology programs and their transition to the Accreditation Council of Graduate Medical Education (ACGME).

We agree that financial factors likely influenced individual graduate medical education (GME) institutions’ decision to pursue accreditation of a particular specialty program over another.

Of note, the authors mention in their letter [1] that two of the former AOA-accredited urology programs that transitioned successfully and five of the ophthalmology programs that did not transition successfully were affiliated with a single university.

Due to the affiliation of these five programs (representing 62.5% of the ophthalmology programs that did not get accredited by the ACGME per the authors) with a single institution, the authors feel that the data regarding the failure of many ophthalmology programs to successfully transition may be misleading.

They seem to suggest that since these five programs were affiliated with one university, it should be considered that only one program did not achieve accreditation rather than five.


To understand this issue, it is important to remember the nature of AOA accreditation, GME, and the Commission on Osteopathic College Accreditation (COCA)-accredited medical schools.

Together, these institutions previously implemented an “Osteopathic Post Graduate Training Institution (OPTI) model.

” The OPTI was a representative body composed of members from AOA affiliate organizations created to ensure that “postdoctoral training programs were operating within approved standards, rules and regulations, and were providing educational training satisfactory to the public interest” [3].

It was commonplace for a DO medical school to be affiliated with various independent GME programs, with a regional accreditor and oversight process, such as the OPTI.


While the authors do not cite which five programs were affiliated with one university, we believe they are referring to the five programs that were affiliated as part of a “Consortium of Osteopathic Residencies in Ophthalmology (CORO)” with a single midwestern university [4].

The CORO was founded in 1989, predating the university’s modern statewide campus system.

CORO featured some shared academic elements, such as didactics and access to a surgical simulator.

However, sharing such resources was and is not unique to the CORO or even the state of Michigan, as Henry Ford ophthalmology is home to a state-of-the-art wet lab and has granted access to neighboring ophthalmology programs.


Despite the five programs sharing a loose affiliation with a single university, these were all separate programs, independently accredited, with distinct program directors and department chairs, without interdependence on one another for accreditation.

The mutually exclusive nature of these five programs was so well understood that when we obtained the program roster from the American Osteopathic Colleges of Ophthalmology and Otolaryngology – Head and Neck Surgery prior to the single accreditation process, the programs were listed separately.

As such, we feel that the data presented in the initial study regarding the percentage of ophthalmology programs gaining ACGME accreditation is an accurate representation.


Interoception is understood as the sensing and representation of signals concern- ing the internal state of the body [1,2].

As a general concept, interoception includes two forms of perception: proprioception (signals from the skin and mus- culoskeletal apparatus) and visceroception (signals from the inner organs such as heart rate, breath and hunger).

Garfinkel & Critchley [3] first emphasized the importance of differentiating between different facets of interoceptive processing, suggesting taking the following into account: objective interoceptive accuracy (IAc; e.g. behavioural testing such as performance on heartbeat perception tests), metacognitive awareness (e.g. confidence-accuracy correspondence) and subjective interoceptive sensibility (e.g. as assessed via self-report questionnaires, e.g. body perception questionnaire).

In a recent study, Garfinkel and co-workers [4] investigated these three levels of processing and could demonstrate that sig- nificant correlations between dimensions emerged only within the subgroup of individuals with higher IAc.

They suggest that the relative balance of accuracy, sensibility and awareness dimensions could explain cognitive, emotional and clinical associations of interoceptive ability.


Referring to the study of Garfinkel et al. [ 4], IAc might be the core ability within the construct of interoception underpinning other interoceptive measures.

Individuals differ substantially in measures of IAc, the ability to consciously per- ceive signals arising from the body.

Measuring a person's ability to perceive and & 2016 The Author(s) Published by the Royal Society.

All rights reserved.

accurately report one's heartbeats at rest is often used to quan- tify these differences [1,5-9].

Other methods exist targeting the gastrointestinal or respiratory system [10-14], but only a few studies have investigated interoceptive abilities across different organ systems [10,15], suggesting that interoceptive abilities are associated across modalities to a certain extent.


The neural substrates of the distinct facets of interoceptive processing are still poorly understood.

The insular cortex plays a major role as a cortical projection area of viscerosensory input [16].

Receiving direct viscerosensory input from the thalamus, the posterior and mid insular cortices are implicated as the primary viscerosensory cortices.

However, other cortical (e.g. ventromedial prefrontal cortex, anterior cingulate cortex, somatosensory cortices) and subcortical (e.g. ventral striatum) regions as parts of the 'interoceptive neural network' also receive ascending interoceptive inputs to support the regu- lation of physiology and behaviour [17].