Enterobacter Sakazakii Blog : Enterobacter Sakazakii Lawyer & Attorney : Marler Clark Law Firm : Enterbacter Sakazakii Blog

In the October 30, 2009, edition of its weekly MMWR publication, the Centers for Disease Control and Prevention (CDC) reported on an investigation in November, 2008, when the Cronobacter sakazakii bacteria was isolated in two different infants. As recognized by the CDC, isolation of this organism from human specimens is rare and makes these cases notable. Cronobacter sakazakii (formerly Enterobacter sakazakii) are rare causes of infant septicemia and meningitis, resulting in death in approximately 40% of cases. Since 1958, 120 cases of Cronobacter sakazakii infection in infants have been reported, an average of fewer than three cases per year worldwide. Powdered infant formula (PIF), which is not sterile, has been implicated repeatedly as a vehicle of Cronobacter infection. This report provides important additional information regarding this elusive pathogen, and updates the CDC’s recommendations regarding safer PIF preparation, storage, and handling.

The Cronobacter sakazakii bacteria were isolated from two non-hospitalized, unrelated infants in November, 2008, in New Mexico. The CDC and FDA investigators determined that the female infant had been infected with Cronobacter sakazakii, and that the male infant had been colonized with Cronobacter sakazakii, without clear evidence of infection. Ingestion of PIF was the only identified risk factor for Cronobacter sakazakii exposure for the two infants. The two infants had consumed the same brand of PIF but had no other common exposures. The female infant had documented Cronobacter sakazakii infection that led to severe brain injury and hydrocephalus. Although a Cronobacter sakazakii organism was isolated from the male infant at autopsy, the role of that organism in the infant's apparent death from SIDS is unknown.

The two infants had consumed the same brand of formula, but their clinical Cronobacter sakazakii isolates had different Pulsed Field Gel Electrophoresis (PFGE) patterns. None of the samples obtained from the home of the female infant yielded Cronobacter sakazakii. Samples taken from the home of the male infant, however, provided positive results for Cronobacter sakazakii. An opened can of PIF yielded a Cronobacter sakazakii isolate with a PFGE pattern that was indistinguishable from the clinical Cronobacter sakazakii isolate from the male infant. Additionally, the vacuum cleaner filter from the home of the male infant also yielded Cronobacter sakazakii, but with a different PFGE pattern than the PFGE pattern isolated in both the male infant and the open PIF can.

The CDC reaffirmed in this report that prior investigations have found Cronobacter sakazakii cultured from prepared formula, unopened PIF containers, and the environment where PIF was reconstituted, clearly implicating PIF as the source of outbreaks. Other than an improperly prepared intravenous nutrition solution implicated in one outbreak, no other clear source of Cronobacter sakazakii infection has been identified to date. Accordingly, the report recommended that preparers should be aware that PIF is not sterile and can contain pathogenic organisms, such as Cronobacter sakazakii. The report also recommended that WHO guidelines for preparation of PIF, including reconstitution with water hot enough to inactivate Cronobacter sakazakii, be adopted, for safer PIF preparation, storage, and handling. In the United States and elsewhere, present recommendations are: to breastfeed infants when possible; to use sterile liquid infant formula in high-risk settings (e.g., neonatal intensive-care units and hospital nurseries); and to adhere to the safest available PIF preparation procedures. Interestingly, the CDC report noted that the manufacture of sterile powdered infant formula, perhaps by using irradiation in combination with other techniques, could prevent infant disease. Finally, the CDC stated that further precautions to prevent extrinsic contamination of PIF are needed, including the engineering of PIF packaging to prevent introduction of contaminated hands, scoops, or other items.

The complete report is accessible at MMWR. 2009:58;1179-1183.

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"Classifications are theories about the basis of natural order, not dull catalogues compiled only to avoid chaos." Stephen Jay Gould, Wonderful Life (1989), 98.

Enterobacter sakazakii, a gram-negative bacillus, is a rare cause of bloodstream and central nervous system infections. In 2007, following extensive study, it was proposed that the original taxonomy of Enterobacter sakazakii be revised, to consist of five new species moved to a new genus, identified as "Cronobacter". (1) A review of the what, the how, and the why the change was first proposed, and why it was eventually approved, provides an insight into the related scientific process of taxonomy at work, involving this notorious neonatal pathogen.

Initially, taxonomy is the science of classifying organisms, identifying and naming species, and organizing them into systems of classification. At least 1.7 million species of living organisms have been discovered, and the list grows longer every year. Ideally, classification should be based on homology, i.e., the shared characteristics that have been inherited from a common ancestor. Until recent decades, the study of homologies was limited to anatomical structures and pattern of embryonic development. However, since the birth of molecular biology, homologies can now also be studied at the level of proteins and DNA. (2)

More specifically, E. sakazakii is a rare, but life-threatening cause of neonatal meningitis, sepsis, and necrotizing enterocolitis. In general, E. sakazaii kills 40-80 % of infected newborns diagnosed with this type of severe infection. (5) E. sakazakii meningitis may lead to cerebral abscess or infarction with cyst formation and severe neurologic impairment. E. sakazakii can cause a variety of infections, though central nervous system infection has been most commonly described. (6) For infants, infection typically manifests through signs of sepsis in the first week of life: irritability or lethargy, temperature instability, and feeding intolerance. Meningitis often produces overwhelming infection that rapidly moves through cerebral hemorrhage, infarct, necrosis, liquefaction, and eventually, cyst formation. (7)

E. sakazakii invasive infections occur more frequently in infants than in older children. (9) The neonate's immature immune system may increase the risk of acquiring an E. sakazakii infection. (10) In a study of E. sakazakii cases over a 47 year period, investigators found that the median age at infection onset was two days and 94% of cases were less than 28 days old. (11)

While the reservoir for E. sakazakii is unknown in many cases, a growing number of reports have established powdered infant formula as the source and vehicle of infection. In several investigations of outbreaks of E. sakazakii infection that occurred among neonates in neonatal intensive care units, investigators were able to show both statistical and microbiological association between infection and powdered infant formula consumption. These investigations included cohort studies which implicated infant formula consumed by the infected infants. In addition, there was no evidence of infant-to-infant or environmental transmission; all cases had consumed the implicated formula. The stomach of newborns, especially of premature babies, is less acidic than that of adults: a possible important factor contributing to the survival of an infection with E. sakazakii in infants. (13)

The first cases attributed to this organism occurred in 1958 in England (Urmenyi and Franklin, 1961). Since then, up to July 2008, around 120 documented cases of E. sakazakii infection, and at least 27 deaths, have been identified from all parts of the world in the published literature and in reports submitted by public health organizations and laboratories. (12)

E. sakazakii used to be previously known as a "yellow pigmented Enterobacter cloacae", until 1980. E. sakazakii was first defined as a novel species in 1980, when it was introduced as a new species based on differences in DNA-DNA hybridization, biochemical reactions, and antibiotic susceptibility. The bacteria was named sakazakii in honour of the Japanese microbiologist, Riichi Sakazaki, when the species was first designated in 1980. (15) Enterobacter sakazakii (E. sakazakii) then became identified as one of sixteen distinct species in the genus Enterobacter, within the Enterobacteriaceae family. (14)

From the beginning, however, many different biogroups were defined as E. sakazakii, with the existence of these divergent geno- and biogroups suggesting that E. sakazakii could in fact represent multiple species. (14) Accordingly, in 2007, a research group clarified the taxonomic relationship among the various E. sakazakii strains, by using sophisticated new means of viewing and analyzing the bacteria. Iverson et al were thus able to distinguish numerous separate species. Their work resulted in the proposal of an alternative classification of E. sakazakii into a new genus, Cronobacter. (16)

The new techniques used by the research group provide clear proof of the substantial advances made in molecular biology, and included f-AFLP, automated ribotyping, full-length 16S rRNA gene sequencing and DNA-DNA hybridization. (14) F-AFLP (fluorescent amplified fragment length polymorphism) is a means to genotype bacteria, by selecting pre-adapted fragments of DNA and amplifying them to easily detectable and accurately sizeable concentrations. Automated ribotyping is a genotyping method that can be used to generate genetic fingerprints of bacterial isolates. Full-length 16S rRNA gene sequencing provides a means to compare a stable part of the genetic code (the 16S rRNA gene) amongst different bacteria. The technique of DNA-DNA hybridization provides genetic comparisons amongst the total genome of two species.

E. sakazakii has thus now been reclassified as 6 separate species in the new genus, Cronobacter, gen. nov., within the Enterobacteriaceae family. The new species are presently Cronobacter sakazakii; C. turicensis; C. malonaticus; C. muytjensii and C. dublinensis; the sixth species is identified simply as genomospecies I, as currently it includes only two representative strains. (19)

The name Cronobacter was appropriately derived from Greek mythology. E. sakazakii constitutes a microbiological hazard in the infant food chain, with historic high mortality in neonates. Accordingly, it was named after the Greek mythological god, Cronos. (17). Cronos was the son of Uranus (Heaven) and Gaea (Earth), being the youngest of the 12 Titans. He eventually became the king of the Titans, and took for his consort his sister Rhea. Rhea bore him a number of children, including Hestia, Demeter, Hera, Hades, and Poseidon. Cronos, however, had been previously warned by his parents that he would be overthrown by his own child. Accordingly, he swallowed all those children. When Zeus was born, however, Rhea hid him in Crete, and tricked Cronus into swallowing a stone instead. Zeus grew up, forced Cronus to disgorge his brothers and sisters, waged war on Cronus, and was victorious. (18)

It was proposed that these species be moved to the new genus, "Cronobacter", in order to facilitate their identification for the diagnosis of infection and the microbiological monitoring of food products. (20) All these species have been linked retrospectively to clinical cases of infection in either infants or adults, and therefore all these species should be considered pathogenic. (21) The correct and more detailed identification of these organisms will improve the understanding of the broader epidemiology of the members of the new genus. 


It is also important, however, that this reclassification of species not be detrimental to health protection measures already in place, and that all these risk organisms continue to be recognized. (22) As the genus Cronobacter is synonymous with Enterobacter sakazakii, current identification schemes developed for E. sakazakii remain applicable for the Cronobacter genus. The reclassification of E. sakazakii to the new genus Cronobacter will not require the modification of dedicated culture-based laboratory isolation and detection protocols. All currently valid laboratory methods will continue to facilitate the recognition of all of the organisms defined within the new taxonomy. (23) Furthermore, the reclassification does not require any change to the regulatory framework currently in place. (24)

REFERENCES:

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Editor's Note: We return to one of the presentations made at last January's Dublin conference on Cronobacter sakazakii.  This one by Dr. Jeff Farber of Health Canada.

Biography: Dr Jeffrey M. Farber gained an MSc and PhD from McGill University, Montreal, Canada, in Medical Microbiology & Immunology and Food Microbiology respectively. He worked as a research scientist and as Division Chief in the Microbiology Research Division before becoming Associate-Director of the Bureau of Microbial Hazards Food Directorate, Health Products and Food Branch, Health Canada where he is currently the Director. Dr Farber`s major interests are Listeria monocytogenes, Enterobacter sakazakii in foods, produce safety, molecular typing, food safety risk assessment and policy. He is a member of the Editorial Board of the International Journal of Food Microbiology; a reviewer for a number of other Journals; and Content Editor for IAFP Report. He is also an Adjunct Professor, at the University of Ottawa where he co-supervise undergraduate and graduate students; sits on a number of graduate student advisory committees; and is a member of thesis committees. Dr Farber is the treasurer of the International Commission on the Microbiological Specifications for Foods (ICMSF), and is a Past-President of the International Association for Food Protection.

Cronobacter sakazakii advice, policy and research in Canada

Although Canada has not had many reported cases of Cronobacter sakazakii, Health Canada has been actively studying this organism since 1998. In 2002, as a result of an outbreak in Tennessee  in the USA, Health Canada issued an advisory to inform Health Professionals in Canada what  measures they could take to reduce the risk to infants, of consuming powdered-infant formula (PIF).

After reviewing the situation at the national level and due to health concerns with powdered formulae and its international trade, in 2003, Health Canada raised this issue at the international level by proposing to revise the Code of Practice for Powdered Formulae for Infants and Young Children at the Codex Alimentarius Committee of Food Hygiene.

Canada volunteered to chair the Working Group that would be developing the Code. Because of the high level of interest in this issue, the Code was completed in four years, which is a relatively short time considering the complexity and politics behind this issue. The Code has contributed to a big improvement in the hygienic conditions in plants manufacturing PIF, resulting in a lower level of product contamination with C. sakazakii. Canada has produced a document detailing Good Manufacturing Practices (GMPs) for Infant Formula in Canada.

The purpose of this text is to establish and document the current GMPs for the production and quality control of infant formula products made for distribution in Canada. Health Canada uses the GMPs as a basis on which to assess the manufacturing information received in pre-market notifications for new or changed infant formulas.

Health Canada does have microbiological criteria for C. sakazakii in PIF; however, they are currently being revised to be more in line with recent Codex thinking. At present, unfortunately, there are no active or passive surveillance systems for C. sakazakii, in Canada, although this has been discussed.

Health Canada has recently adapted and condensed FAO/WHO guidelines to develop a draft guidance document on the preparation and handling of PIF in home and hospitals/care settings, which outline requirements for parents, caregivers, and staff in hospitals and day-care centers. The guidance document can be used to educate parents, caregivers and staff in hospitals and day-care centres, on the potential hazards associated with PIF.

Health Canada’s Bureau of Microbial Hazards conducts research focussed at examining the ecology, biology and pathogenesis of the organism. Some of the research projects include specific aspects of molecular typing, virulence studies involving animal models, as well as in-vitro tissue culture work to examine adhesion and invasion. Collaborative research is also being done with the National Research Council, using NMR and mass spectroscopy to reveal the structure of the O- polysaccharide of the various Cronobacter species.  For more, see his Powerpoint.

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From news services in Seoul comes world that a South Korean baby formula is contaminated with Enterobacter sakazakii, the baceria that can cause meningitis in infants.

South Korea's National Veterinary Research and Quarantine Service (NVRQS) said Enterobacter sakazakii bacteria was found in 695kg of powdered milk made by Maeil Dairies Co. June 17. The discovery was made during regular sample testing by the agency under the food and agriculture ministry.

The government agency said the 53,460 individual 13g packages were contaminated overall, although none had reached the retail market.

Enterobacter sakazakii or Cronobacter poses particular risks to babies under six months old or weighing under five and half pounds. However, it poses no threat if mixed with boiling water exceeding 158 decrees.

"Investigators are trying to find the cause of the contamination that may have been related to the manufacturing process or the ingredients used," an NVRQS official said.

He said all products suspected of being tainted with the bacteria are currently in a holding area and will be destroyed, with Maeil ordered to take steps to prevent a recurrence.

The products contaminated were all disposable packages of the company's Premium Goong 1 baby formula. Larger cans containing the formula were not tainted with the bacteria.

According to financial websites: Maeil Dairy Industry Co., Ltd. with annual sales in the $1 billion range produces baby foods, beverages, yogurts, and soybean milk products. It also offers milk, fermented milk, cheese, nutritional meals for the pregnant, and oil products. In addition, the company imports and supplies chocolates, olive oil, and grape seed oil. Its products are used in hotels, restaurants, bakeries, and coffee franchises. Maeil Dairy Industry Co., Ltd. offers its products through contracted distributors. The company exports its products to Saudi Arabia, Jordan, Syria, Sudan, Egypt, the United Arab Emirates, China, Japan, Hong Kong, Macao, Singapore, Vietnam, Bangladesh, Afghanistan, the United States, Guam, Canada, Mexico, and the Russian Federation.
 

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The Codex Alimentarius Commission (CAC) adopted 30 new international standards and guidelines to improve food safety and protect consumers, including some that impact powdered infant formula.  The Codex changes came during a just ended week-long meeting.

The Commission adopted criteria for salmonella and other bacteria in powdered follow-up formulae for children six months of age or older and for special medical purposes for young children.

A bacterium of special concern is E. sakazakii, for which Codex adopted specific criteria for powdered formula for infants (0 to 6 months) in 2008. The Commission decided that in countries with particular risk for E. sakazakii from consumption of follow-up formulae (i.e. countries with substantial populations of immunocompromised babies) similar criteria for E. sakazakii could be introduced for follow-up formula as for powdered formula for infants.

Follow-up formulae should only be used for the intended target population. Unfortunately, they are often consumed by babies younger than six months of age. The standard stresses the need to address such product misuse issues through education campaigns and training. 

Codex, established in 1963, has 181 member states plus the European Community.  It is an offshoot of the United Nations.

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1st Annual Canadian Perinatal & Pediatric Nutrition Conference

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Editor's Note:  We continue presentations from the international Cronobacter conference held earlier this year in Ireland.  In this segment, we hear from the USA's Dr. Keith Lampel from FDA.

Biography: Dr. Keith Lampel is director of the Division of Microbiology at the U.S. Food & Drug Administration (FDA). He joined the FDA as a research microbiologist in 1987 after five years as a senior staff fellow at the National Institutes of Health, National Institute of Neurological Disorders and Stroke.
Dr. Lampel received his PhD in Microbiology from the University of Miami and was a postdoctoral fellow at the State University of New York at Stony Brook.
At FDA today, he is responsible for developing bacteriological and molecular-based methods to isolate, detect, and subtype foodborne pathogens. Dr Lampel is also the Editor of FDA’s Bacteriological Analytical Manual and serves as the FDA’s expert on the detection and isolation of the foodborne bacterial pathogen Shigella.
Other professional activities include serving on several editorial boards; NIH and USDA study panels, and ad’hoc review panels for several journals and extramural grant programs. He also serves as an adjunct professor at the University of Maryland and Uniformed Services University of the Health Sciences, and is a member of several PhD dissertation committees.

Summary - Development of an FDA/AOAC standard method for detection of Cronobacter

Although the number of incidences of illnesses caused by the ingestion of the bacterial pathogen Cronobacter (Enterobacter sakazakii) has not been as dramatic as other foodborne pathogens, a need remains for a robust isolation method to recover this microbe from powdered infant formula (PIF).
The current method described on the FDA website was developed in response to one such incident. Although C. sakazakii was a rather novel pathogen in an unusual food matrix, a method was devised quickly and applied to PIF samples. Unfortunately, this method requires multiple steps and at least 3-4 days for complete analysis of PIF for isolation and confirmation of C. sakazakii from the formula sample.
The revised method, however, includes a bacteriological enrichment and isolation protocol as well as the integration of a PCR-based assay. As for the bacteriological application, plating follows one-step enrichment on chromogenic agar(s) for presumptive identification of C. sakazakii. Suspected colonies are confirmed by either biochemical analysis or a real-time PCR-based assay. Therefore, isolation and identification of E. sakazakii from PIF is markedly improved and can be accomplished in 24-28 hrs.

Dr. Lampel's POWERPOINT can be found here

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Editor's Note: Another of the presentations follows from last January's 1st International Conference on Cronobacter (Enterobacter Sakazakii) held at University College Dublin.  Dr. Han Joosten from the Nestle Research Center in Switzerland addresses the standard method for detection of Cronobacter.

Biography: Dr. Han Joosten is a Senior Scientist at the Nestle Research Center in Lausanne, Switzerland. At Nestle since 1996, he is responsible for providing scientific guidance on various research projects and early identification of emerging microbiological safety issues. He also provides advice to the business and quality management on analytical methods, hurdle technology, safety assessments and HACCP.
After finishing his studies at the University of Nijmegen in the Netherlands in 1983 Joosten worked five years at NIZO Food Research on the formation of biogenic amines in cheese, obtaining his PhD degree from the University of Wageningen on this subject.
From 1989 to 1991 he worked as a postdoc at the Autonomous University of Madrid on molecular characterization of African Swine Fever Virus. After this he headed the microbiological laboratory of Coberco Research in Deventer, the Netherlands and moved back to Spain in 1994 where he worked for two years at the National Instititute for Agricultural and Food Research (CIT-INIA) in Madrid on a bacteriocin-producing Enterococcus strain.

Summary - Development of a CEN-ISO horizontal standard method for detection of Cronobacter
The availability of a reliable and internationally accepted reference method for detection of Cronobacter in powdered infant formula is an essential tool to verify compliance with regulatory requirements by public health authorities and manufacturers. ISO-TS 22964:2006 was developed as a temporary solution for this purpose, but shortly after being issued it was decided to prepare a full-fledged horizontal CEN-ISO standard.
A summary will be given of the work done thus far, in particular with respect to the modifications that are envisaged to address the main shortcomings of TS 22964:

• The scope will be extended to all types of powdered infant formula (incl. soy- based) and infant formula ingredients.
• It will take into account the latest taxonomical revisions (e.g. definition of the genus Cronobacter and phenotypically related species)
• It will no longer use yellow pigment production as a confirmation criterion
• The enrichment broth (mLST) and chromogenic isolation agar (ESIATM) are too selective and need to be replaced by media that will also allow the detection of strains that are very susceptible to commonly used inhibitors of gram positive microorganisms.
• The main performance characteristics of the new standard will be determined

Based on the results obtained during an extensive comparative/collaborative trial a method based on the utilization of Cronobacter Screening Broth (CSB) in combination with modified DFI agar appears to be the most suitable procedure to be adopted in the new standard.

His POWERPOINT can be found here.

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 Editor's Note: This is another report on the presentations that were made in Dublin earlier this year at the 1st International Meeting on Cronobacter (Enterobacter Sakazakii).  In this segment, we hear from Dr. Patrick Druggan, Oxoid Ltd., Thermo Fisher Scientific, Basingstoke, Hampshire RG24 8PW, United Kingdom.

Biography: Patrick Druggan  is Principle Scientist, Oxoid Thermofisher, Basingstoke, UK.

He received an Honours BSc in Food Science from the University of Strathclyde, Glasgow, UK. He has worked in the diagnostics industry for 22 years. He designed his first chromogenic medium in 1989 while working at Gibco.

Patrick studied part-time for his Ph.D. at the Pharmacy Department of University of Brighton, UK. His thesis was on improvements in the resuscitation of heat-injured Salmonella species from processed food samples.

He synthesized a number of autocytotoxic compounds that could be used during pre-enrichment to inhibit competitive microflora while allowing injured Salmonella spp. to resuscitate and grow.

This invention lead to the development of Inhibigens.™ His skills in chemistry and microbiology have allowed him to design a number of successful rapid biochemical tests and chromogenic culture media, including Druggan-Forsythe-Iversen Agar for the isolation of Cronobacter spp. 

Summary – - Culture media for isolation and detection of Cronobacter species

In 2001 a pre-term infant died of meningitis caused by Enterobacter sakazakii (Cronobacter spp.).  Infant formula milk (IFM) was implicated as a potential source of the infection.

The Food and Drug Administration (FDA) independently develop a method for enumeration of this emerging  pathogen in IFM using culture collections from national bodies that have later been shown to be poorly defined.

This method was introduced in 2002 and has regulatory standing for the import of IFM and skimmed milk powder in to the USA and a number of other countries. The FDA method is a modification of the procedure for the detection of Enterobacteriaceae, with the addition of yellow pigmentation of colonies for presumptive identification of Cronobacter spp.

It should be remembered that the FDA method was developed in a short time due to a public health concern, and this would have put a time constraint and significant pressure on those working on Enterobacter sakazakii (Cronobacter) to get a working method in the field as soon as possible. The FDA method has been shown to have a sensitivity of around 50 percent and a specificity of around 70 percent. Only 75 percent of Cronobacter strains phenotypically express yellow pigmentation, and the low specificity of the method coupled with the recommendation that only five presumptive Enterobacteriaceae colonies are tested from Violet Red Bile Glucose Agar (VRBGA) may explain the poor sensitivity of the method.

Assuming the prevalence of Cronobacter spp. in IFM is around 2 percent, the FDA method will fail to detect around 50 percent of batches contaminated with Cronobacter, while around 95 percent of rejected batches will not contain this organism.

This high rate of failure has lead many stakeholders to question the usefulness of the FDA method. This presentation reviews developments in culture media since the release of the FDA method in 2002, with specific emphasis on media that have improved the specificity of methods for Cronobacter spp. The unique phenotypic trait of this emerging pathogen that aid and hinder design of methods is discussed.  

POWERPOINT is here.

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