Corynebacterium Ulcerans Diphtheria: an Emerging Zoonosis in Brazil and Worldwide

The article is a literature review on the emergence of human infections caused by Corynebacterium ulcerans in many countries including Brazil. Articles in Medline/PubMed and SciELO databases published between 1926 and 2011 were reviewed, as well as articles and reports of the Brazilian Ministry of Health. It is presented a fast, cost-effective and easy to perform screening test for the presumptive diagnosis of C. ulcerans and C. diphtheriae infections in most Brazilian public and private laboratories. C. ulcerans spread in many countries and recent isolation of this pathogen in Rio de Janeiro, southeastern Brazil, is a warning to clinicians, veterinarians, and microbiologists on the occurrence of zoonotic diphtheria and C. ulcerans dissemination in urban and rural areas of Brazil and/or Latin America.


INTRODUCTION
Diphtheria is a disease with acute evolution that shows local and systemic manifestations.It remains an important cause of morbidity and mortality on different continents, even in countries with child immunization programs. 4,35,58,82,92The classical forms of diphtheria are caused mainly by Corynebacterium diphtheriae, which produces diphtheric toxin (DT), and are characterized by the presence of a grayish pseudomembrane at the infection site, due to the effects from multiplication of this bacillus and the host's immune response. 40,65,114DT is a potent exotoxin of protein nature that has the capacity to act on all tissues, with special tropism for the myocardium, nervous system, kidneys and suprarenal glands. 40,82,93Only the samples infected by specifi c bacteriophages that carry the gene tox are toxinogenic. 33otection against DT can be acquired through vaccination with diphtheric toxoid (e.g.a The level of immunity diminishes at the end of childhood and adolescence, depending on the vaccination calendar and the reservoir of C. diphtheriae in the population. 35,77Diphtheria may affect partially immunized individuals, independent of age, race or sex.Partial decline in immunity, allied with lack of vaccine boosts and lower exposure to the a Secretaria de Saúde do Estado do Rio de Janeiro.Proporção de casos e óbitos por difteria segundo faixa etária, 1978-2002.Rio de Janeiro: Assessoria de Doenças Imunoprevisíveis; 2002. bacillus, contribute towards sporadic occurrences of epidemics. 23,34,69,77 today's era of vaccination, the various changes observed in the epidemiology of diphtheria 14,35,69 can be partially explained by the prevailing low levels of diphtheric antitoxin in the adolescent and adult population. 15,29In the 1990s, in Eastern Europe, the greatest epidemic of diphtheria recorded since the start of mass vaccination occurred, accounting for 80% of the cases notifi ed worldwide.Most of the cases and deaths occurred among adults infected by the epidemic clone of C. diphtheriae subsp.gravis, which still circulates in Russia, Latvia, Lithuania and Belarus. 89,90,112,113 addition to changes in age group, diphtheria cases without pseudomembranes and cases of invasive infection of a variety of origins have been observed in individuals who had previously undergone vaccination.These cases of invasive infection such as endocarditis, osteomyelitis, arthritis, pneumonia and renal abscesses have been predominantly correlated with atoxigenic samples of C. diphtheriae subsp.mitis. 17,24,48,53,66,68e World Health Organization (WHO) has created the Disability-Adjusted Life Year (DALY) index, in which each unit represents one year of healthy life lost through disease, hospitalization or even death (taking into account the life expectancy in each country), per 100,000 inhabitants.The 2002 data on the mortality and incidence of diphtheria correlated the following regions with greater DALY indexes: Central America [Haiti (> 50 DALY units) and the Dominican Republic], Sub-Saharan Africa [Democratic Republic of the Congo and Serra Leone] and Asia [Nepal and Myanmar] (> 15 DALY units).b The current epidemic that has been occurring in India presents DALY > 7 and vaccination coverage less than 80%.Only 56% of the children are protected against diphtheria (≥ 0.1 UI/ml). 4e countries of South and North America, Western Europe and Oceania present DALY indexes of less than one.b In Brazil, the national immunization program was established at the start of the 1970s.c,d According to data from the Brazilian Ministry of Health, vaccination coverage now reaches 99% of the child population.e,f,g,h Over the period 2008/09 alone, 89 diphtheria cases were confi rmed.In 2010, in three municipalities in the state of Maranhão, where vaccination coverage reaches 56%, 27 diphtheria cases were confi rmed, with two deaths, and the majority were in children with complete vaccination schemes.The possibility of underreporting of cases in Brazil and other developing countries cannot be dismissed: there may be unawareness among the population regarding the need to seek medical care and diffi culties in obtaining clinical-laboratory diagnoses in cases of diphtheria, especially when patients are partially protected against the action of DT.
Starting in the middle of the 1980s, the number of diphtheria of zoonotic nature caused by C. ulcerans increased in different countries.In England, the number of diphtheria cases due to C. ulcerans exceeded the number reported with the classical etiological agent, C. diphtheriae. 5,76,96,107,109,113,115Although toxigenic C. ulcerans has now been recognized in several industrialized countries as an emerging pathogen, its capacity to cause disease in humans, including among the inhabitants of urban centers, is still often neglected. 19,70,108us, the present review had the aim of analyzing aspects of the emergence of C. ulcerans as an etiological agent for zoonotic diphtheria in Brazil and other countries that carry out triple bacterial vaccination (DTP), against diphtheria, tetanus and pertussis, and other combinations containing the diphtheric component.

METHODOLOGICAL PROCEDURES
Studies and surveys on zoonotic diphtheria were gathered by searching in the Medline/PubMed and SciELO databases.The search terms used were: "diphtheria", "Corynebacterium ulcerans", "zoonotic diphtheria", "laboratorial diagnosis", "diphtheria clinical cases", "molecular epidemiology" and "infected animals", covering the period from 1926 to 2011.Studies focusing on descriptions of diphtheria cases due to C. diphtheriae that occurred outside of Brazil were excluded.In addition, articles available in electronic format in Portuguese and English focusing on the diphtheria situation in Brazil, laboratory procedures and serum levels of diphtheric antitoxin antibodies in adults, were analyzed.

MICROBIOLOGICAL AND CLINICAL CHARACTERISTICS
The genus Corynebacterium belongs to the group of irregular Gram-positive rods, which grow aerobically and are immobile, non-sporulating and catalase-positive.Over the last few years, more than 60 species of corynebacteria have been described, and many of them are related to colonization and/or infection in humans and animals.Phylogenetic analysis on subunits of 16S rRNA sequences has made it possible to identify C. ulcerans, C. diphtheriae and Corynebacterium pseudotuberculosis as distinct species that are potential producers of DT. 57,91 C. ulcerans and C. pseudotuberculosis are capable of hydrolyzing urea, which presumptively differentiates them from the type species of the genus, i.e. C. diphtheriae.
The species C. ulcerans, which is gelatinase-positive and nitrate-negative and was described by Gilbert & Stewart in 1926, can produce several clinical conditions, depending on the type and quantity of toxins secreted. 8,36Many studies have investigated pathogenicity mechanisms and DT production in samples of C. diphtheria, 13,47,49,69,73,101 but little has been documented about the virulence factors of C. ulcerans.The virulence mechanisms described for C. pseudotuberculosis include toxic lipids associated with the cell wall, which may mediate bacterial to attack by phagocytes. 42Currently, a phospholipase D (PLD) exotoxin is considered to be the main virulence factor produced by C. pseudotuberculosis during cases of infection.PLD presents sphingomyelinase activity and may locally increase the vascular permeability, thereby contributing towards dissemination of the pathogen in the host's tissue. 3,50,72C. ulcerans is also capable of producing PLD, as well as DT. 51ncern regarding the potential for C. ulcerans to emerge as a pathogen capable of carrying the DT-coding bacteriophage, and therefore capable of causing conditions of classical diphtheria, is not new.Studies have shown that some bacteriophages are selective regarding C. ulcerans and do not induce DT production. 45However, it has also been observed that the bacteriophage β has the capacity to infect samples of C. diphtheriae, C. pseudotuberculosis and C. ulcerans, thereby constituting a risk of emergence of conditions of diphtheria caused by this species. 1,38,95,99Variability of the capacity to produce DT, between samples of C. ulcerans, has already been documented. 39,116The majority of clinical cases in humans and animals have been correlated with DT-producing strains (Tables 1 and 2). 11,18,56,62e following DT effects are produced by C. ulcerans: frequent occurrences of nose bleeding during infections; skin lesions that mimic typical cutaneous diphtheria; lesions of the tracheobronchial tree, with pseudomembrane; hemorrhage; compromised cervical lymph nodes; and cell death due to apoptosis. 8,17,63,111,115Occurrences of necrosis, mucosal ulceration and clinical syndromes in the lower respiratory tract, such as pneumonia and granulomatous pulmonary nodules, have been attributed to production of DT and PLD. 16,44,52,97

EPIDEMIOLOGICAL CHARACTERISTICS
Classically, the species C. ulcerans has been described as the etiological agent of a variety of infectious conditions in animals, especially mastitis in cattle.The fi rst cases of infection in humans have usually been associated with consumption of unboiled or unpasteurized milk and derivatives, along with rural workers who are asymptomatic carriers of C. ulcerans in the nasopharynx.This shows the capacity of this pathogen to circulate between human and animal hosts and cause diphtheria of zoonotic nature. 2,7,43,46,52In a general manner, diphtheric infections caused by C. ulcerans have been correlated with patients who have been partially immunized with diphtheric toxoid, negative results from investigating carriers and contacts and indeterminate infection sources. 108 presented in Table 1, in addition to conditions with characteristics similar to respiratory and cutaneous diphtheria, samples of C. ulcerans have also been correlated with other clinical conditions in humans, such as sinusitis, tonsillitis, pharyngitis, pneumonia and peritonitis.The growing number of cases of infections due to C. ulcerans in cats and dogs since 2006 emphasizes the importance of expanding the knowledge of the epidemiological aspects of this emerging zoonosis.Cases of infection due to C. ulcerans in various animals species such as monkeys, squirrels, otters, orcas, camels, lions, dogs, cats, pigs, goats and cattle have been described in the international literature (Figure 1 and Table 2). 1,17,18,55,62,96,103ong the 37 studies relating to infections in humans, it was possible to determine the sex of 37 individuals and the age in 32 cases.The majority of the cases (n = 24; 65%) and deaths (n = 5; 71%) were observed in females.Interestingly, most of the infections in animals due to C. ulcerans have also occurred in females (mastitis).Wagner et al 113 (2010) conducted a survey of cases of infections due to C. ulcerans and C. diphtheriae in England between 1986 and 2008, and also found that the cases were predominantly in women (76%), as were the deaths (75%).The mean age of the individuals with C. ulcerans (38 years) was higher than the mean age of those with C. diphtheriae (15 years).In addition, Table 1 shows that the individuals with C. ulcerans were older (mean of 53 years), with a large number over 60 years, which contrasts strongly with the age profi le of          As presented in Table 1 and Figures 1 and 2, C. ulcerans has been a growing cause for concern among the public health authorities in many countries: England, 25,113 France, 1 Germany, 100 Netherlands, 108 Italy, 110 Switzerland, 39,56 Denmark, 75 Japan, 44 Canada 17 and the United States. 12,13,107Only fi ve cases of human infection were documented prior to the 1990s, and these were in England and Denmark.Many cases of human patients who had not presented risk factors associated with infections due to C. ulcerans (such as consumption of untreated milk or contact with farm animals) started to be reported from 1990 onwards.
Therefore, the risk of transmission among small animals and among humans has become a cause for concern. 1,17,18,55,62,70,96,107 addition to cattle and goats, dogs and cats have also become responsible for transmitting C. ulcerans to human hosts.In two cases of diphtheria caused by C. ulcerans that were described in France (2005 and 2006), the source of infection was the pet dog. 1,62ibotyping tests were also been conducted in order to determine the genetic relationship between the microorganisms isolated from these animals and those in humans.In the United Kingdom, it was observed that toxigenic samples isolated from cats with bilateral nasal discharges belonged to the same ribotypes of C. ulcerans that were isolated from infected humans, thus showing these animals' capacity to act as pathogen reservoirs. 18,104Likewise, it was observed that a sample of C. ulcerans isolated from humans in Italy belonged to the same ribotype found in samples isolated in England. 110though C. diphtheriae is classically considered to be a human pathogen, cases of infection due to C. diphtheriae in cats (severe otitis) and a horse (purulent skin lesions) were reported in 2010, 41,64 thus suggesting that zoonotic potential was also emerging with regard to C. diphtheriae (Table 2).
Transmission of C. ulcerans between humans has still not been demonstrated, but several researchers have recommended that infected patients should be isolated. 2,7,11,43,60,70,107In the fi rst and only clinical case of human infection due to C. ulcerans notifi ed in Brazil, 70 the patient's vaccination history and the source of infection were unknown.A few days after the patient died, one of the healthcare professionals involved in the case presented a condition of pharyngitis consequent to exposure to the pathogen.He immediately started antibiotic therapy with erythromycin, before material was collected for culturing and identifi cation.Although the culture presented a negative result, the patient presented signs of myocarditis in clinical cardiological examinations, including echocardiograms and repeated electrocardiograms.
The causes contributing towards the increase in the number of cases of infection due to C. ulcerans in humans and animals have still not been fully elucidated.
Given that similar ribotypes have been observed in human and animal infectious processes, it is possible that some samples of C. ulcerans may present additional virulence factors resulting from selective pressure, associated with unfavorable socioeconomic conditions, especially in overpopulated areas in which inadequate sanitary and hygiene conditions prevail.In addition, in some countries like the United States, the number of domestic animals living inside homes and circulating in areas such as kitchens and bedrooms, is very high, i thus favoring the process of transmission of various agents and zoonoses to humans, possibly including C. ulcerans. 20

PRESENT SITUATION IN BRAZIL AND EMERGING COUNTRIES
As illustrated in Figure 1, the number of cases notifi ed in emerging countries is very small.In Latin America, cases in animals have been described in Argentina 74 and Brazil, 19 but in humans, only in Brazil 70 (Tables 1 and    America Central America and Caribbean  2).There are no records in the literature of isolation of samples of C. ulcerans in India, China, Oceania or the entire African continent.The hypothesis that circulation of this pathogen really occurs in all continents also needs to be put forward, and that there may be underreporting of cases because of lack of availability of laboratories qualifi ed to isolate and identify the pathogen.Other than the United Kingdom, few countries present established laboratory routines and require compulsory notifi cation of human infection due to C. ulcerans and isolation of atoxigenic samples.
In August 2010, the Brazilian Ministry of Health issued a warning j about the diphtheria situation in this country.This document gave information about changes to the clinical-epidemiological profi le of the disease, such as the absence of pseudomembranes, deviation of the age group, occurrences of zoonotic diphtheria and circulation of C. ulcerans and C. diphtheriae in Brazil.Other points that were highlighted included: adoption of the same control measures as recommended for the species C. diphtheria, in confi rmed cases of diphtheria due to C. ulcerans; and notifi cation, treatment and monitoring of all suspected cases of diphtheria, with isolation of non-toxin-producing strains, by healthcare professionals.
In Brazil, a single human case of fatal infection caused by C. ulcerans was described in 2008, 70 and subsequently (2010), a dog was found to be an asymptomatic carrier.Both of these cases were in the metropolitan area of the city of Rio de Janeiro (Southeastern Brazil). 19n summary, an elderly women presented several skin lesions on her legs, covered by pseudomembranes, and she died of cardiorespiratory complications despite antibiotic therapy serum therapy.The microorganism isolated from the patient's lower respiratory tract, which produced an exotoxin presenting differences in subunit A of the molecule, was shown to be resistant to the antimicrobials penicillin G (MIC 0.19 mg/l) and clindamycin (MIC 1.5 mg/l).Variations in sensitivity profi les had previously been observed by other authors. 15,37n the same way as found on other occasions, like in the United States, 11,107 Switzerland, 39 Germany, 54,111 England 8,63,105 and Japan, 59,61,76,96 the origin of the infection was not elucidated (Tables 1 and 2).
According to Pesavento et al 84 (2007), several diseases are reemerging.Hypotheses raised to explain these occurrences include the sanitary status of the population, migrations, adaptation by pathogens to new environmental conditions and factors, among others.According to those authors, animal shelters present many conditions that predispose towards this phenomenon: stress, immunosuppression, overpopulation, high Such fi ndings were the motivation for conducting a microbiological survey among apparently healthy dogs at an animal shelter.Out of a group of only 60 dogs from which nasal and ocular secretions and skin lesions were analyzed, one female dog that was an asymptomatic carrier of C. ulcerans was detected. 19ndings of C. ulcerans in the environment, infecting and/or colonizing humans and/or dogs 19,70 suggests that the scarcity of epidemiological data in Brazil may be related to clinicians' and microbiologists' lack of attentiveness regarding the possibility of infection and a lack of awareness of the clinical and microbiological characteristics of infectious processes caused by C. ulcerans.Moreover, there are no routine procedures that enable isolation and identifi cation of this pathogen in clinical laboratories.

BACTERIOLOGICAL DIAGNOSIS: TRIAGE SCHEME
29]35 In emerging countries, in which the incidence of diphtheria remains relatively high and the vaccination coverage continues to be insuffi cient, laboratory support should be implemented in the light of the recurrent defi ciencies in this segment. 30 is essential to have accurate, rapid, economical and easily performed triage, with the capacity to make a presumptive diagnosis of diphtheria cases caused both by C. ulcerans and by C. diphtheriae, in the majority of Brazilian laboratories.This would have the aim of reducing the underreporting that results from false negative laboratory results.
In Figure 3, the proposed algorithm enables isolation of Gram-positive rods that are potential DT producers, using culturing media (blood agar and tellurite chocolate agar) and biochemical tests routinely used in clinical bacteriological laboratories, i.e. deoxyribonuclease (DNAse) activity, urea hydrolysis and the reverse CAMP reaction using a beta-lysine-producing strain of Staphylococcus aureus.If it is possible to use Tinsdale medium, the samples of cystinase-negative Grampositive rods can be discarded.Cystinase-positive and  H 2 S-producing samples are identifi ed through the presence of a brown halo around the colony. 30,36sitive results from the DNAse test 88 suggest that C. diphtheriae and C. ulcerans are present.Unlike C. diphtheriae, samples of C. ulcerans are ureasepositive and undergo the reverse CAMP reaction. 35n rare occasions, samples of C. pseudotuberculosis that are potential DT producers may be isolated.These present negative results from DNAse tests and positive results in the cystinase, urease and reverse CAMP tests.Cystinase-negative and DNAse-negative samples should be considered to be "other coryneform bacteria".
In addition to conventional biochemical tests, the semiautomated API Coryne system (BioMérrieux, Lyon, France) is also capable of identifying these pathogens, including the atypical sucrose-fermenting samples of C. ulcerans and C. diphtheriae that are often isolated in Brazil. 33,67boratories that do not have any resources available for carrying out toxigenicity tests should send the strains for complementary analyses to the Central Public Health Laboratory of their region and/or the National Reference Laboratory and the Collaborating Center for diphtheria.Laboratories that do have resources available for conducting molecular techniques capable of identifying C. diphtheriae and C. ulcerans and determining the presence of the tox gene 9,79,80,86,87 should also send the strains for epidemiological control procedures, to the same laboratories, which have this capacity.
Considering the short incubation period of this disease (between one and six days), it is recommended that material for investigating C. ulcerans and C. diphtheriae should be collected from nasal and oropharyngeal mucosa and skin lesions of people and animals that have been in contact with the case (suspected or confi rmed) over the last 10 to 14 days.

ROLE OF DIPHTHERIC TOXOID IN PREVENTION
The concentration of protective antibodies in adult individuals decreases by 10% every year. 98The importance of vaccination booster programs has been proven in Finland. 78Data on vaccination coverage among adolescents and adults in Brazil are scarce in the literature. 15,20,22,85,102In the city of Rio de Janeiro, only 30% of the adults are completely protected against the action of DT, 15 similar to what has been observed in other countries like Turkey. 10 These data emphasize the importance of applying reinforcement doses of diphtheric toxoid every ten years, in order to avoid decreased antibody levels in the population. 10,20wever, the effi cacy of diphtheric toxoid against the zoonotic diphtheria caused by C. ulcerans still remains unknown.The studies listed in Table 1 showed that the majority (approximately 75%) of the cases of zoonotic diphtheria have occurred in adult patients who had been fully or partially vaccinated with diphtheric toxoid.
There are still many issues that require better assessment, not only in relation to clinical-laboratory diagnosis, but also in relation to treating and preventing diseases caused by C. ulcerans.Likewise, better comprehension of the molecular epidemiology and characteristics of C. ulcerans, along with the toxin produced (which resembles the DT of C. diphtheria) is needed.
Recent studies have revealed differences in nucleotide sequences between the tox genes of C. ulcerans and C. diphtheriae, and between the tox genes of different samples of C. ulcerans.Some samples isolated from patients presenting conditions of extrapharyngeal infection have presented differences in DT sequences, predominantly in the domains of translocation and adherence. 9,70,80,99,109ese facts may contribute towards situations in which individuals vaccinated with diphtheric toxoid or undergoing serum therapy do not present full protection against infections caused by C. ulcerans.Even if it is considered that the diphtheric toxoid may have a protective effect against diphtheria caused by C. ulcerans (i.e. through the presence of attenuated clinical symptoms), it should be remembered that the vaccination only presents the action of DT and probably does not impede colonization by toxigenic corynebacteria.There is agreement among researchers that, in the absence of a proven vaccine against C. ulcerans, diphtheric toxoid remains a reasonable alternative, especially in cases of convalescence.On the other hand, it is also believed that there is a scarcity of evidence to show that vaccination with diphtheric toxoid, even when kept up-to-date, would impede zoonotic diphtheria or other diseases caused by C. ulcerans. 94,107

FINAL REMARKS
The circulation of C. ulcerans in many countries, added to the recent cases of infections in humans and animals described in Brazil, make it possible to raise the hypothesis that zoonotic diphtheria is occurring and C. ulcerans is circulating in urban and rural regions of Brazil.There is thus a need to improve the adequacy of the protocols for microbiological diagnosis of both C. ulcerans and C. diphtheriae, using alternative triage methods that could be implemented in public and private-sector laboratories.Implementation of epidemiological and laboratory-based surveillance may contribute towards increasing the number of confi rmed cases of classical and zoonotic diphtheria in Brazil.
positive for C. ulcerans.Negative investigation on in rural area/investigation of C. ulcerans in milk samples: 2 cats and 5 dogs (2 dogs positive) England (2010)105

Monkey ( 1 106 Camel 62 Dog
from 56% of the skin lesions after implantation and from 3% of the oropharyngeal samples Spain (2000) as isolated from 50 individuals were isolated from the cats, thus suggesting that these animals might have acted as reservoirs for blood and lung tissue of 2 orcas (male and female) kept in the same aquarium France (2005) Adult recovered from condition of classical diphtheria.The pathogen was isolated from the animal's skin lesions, tonsils and nostrils Argentina (classical diphtheria.Strain isolated from human was identical to one from pig.Investigation of 3 human contacts was negative To be continued

Figure 1 .
Figure 1.Worldwide distribution of notifi ed cases of infection due to Corynebacterium ulcerans in humans (Stars) and in animals (Triangles), 1972 to 2010.

Figure 2 .
Figure 2. Distribution in different countries of cases of human infection due to Corynebacterium ulcerans reported in the literature available, 1984 to 2010.

Table 1 .
Cases of infections due to Corynebacterium ulcerans in humans reported internationally in the literature.

Table 2 .
Cases of infections due to Corynebacterium ulcerans and Corynebacterium diphtheriae in animals reported internationally in the literature Country (year)

Table 2
, high turnover of animals, malnutrition and indiscriminate use of antibiotics, thereby becoming a risk factor for the human and animal populations.