Pathological profiles of systemic infections by Panton-Valentine leukocidin-positive, methicillin-resistant Staphylococcus aureus strains in a murine model.

AIMS: Staphylococcus aureus is one of the most common pathogens in hospital environment and community. Panton-Valentine leukocidin (PVL) production is clinically associated with skin abscesses, soft tissues infections, bacteraemia and sepsis. This study aimed to investigate the effects of the presence of genes lukF/S-PV coding for PVL, in histological and haematological features during systemic infection, using a Swiss mice experimental model. METHODS AND RESULTS: Experiments were performed using 25 mice distributed into five experimental groups, intravenously inoculated with 50 µl suspensions at density 1·0 × 107  CFU per ml of strains: methicillin-susceptible (MSSA) and pvl-negative strains isolated from nasal colonization; MSSA pvl-positive strains isolated from nasal colonization; methicillin-resistant (MRSA) and pvl-positive strains isolated from peripheral blood of a patient with severe pulmonary infection; and a MRSA pvl-positive strains isolated from a peripheral blood culture of a patient with bacteraemia. Haematological analysis was performed at 24, 48, 72 and 96 h post-infection. Morphoanatomy and histopathological analyses were performed at 96 h post-infection. For all S. aureus strains tested, the capability of intravenous dissemination and survival into mice tissues was demonstrated. Inflammatory processes at different levels were related to the presence of pvl genes, and included alterations in the format, size and colour of the organs. Staphylococcus aureus pvl-positive strains were detected in greater numbers in the organs of the infected animals. CONCLUSIONS: The pvl-positive strains isolated from blood cultures were capable to induce the greatest modifications in both haematological and histopathological profiles, and seemed to aggravate the systemic infections. SIGNIFICANCE AND IMPACT OF THE STUDY: These findings are valuable in characterizing infections caused by S. aureus in humans and murine.

Genomic analysis of a novel nontoxigenic Corynebacterium diphtheriae strain isolated from a cancer patient.

The genome of a novel nontoxigenic Corynebacterium diphtheriae, strain 5015, isolated from a patient with adenoid cystic carcinoma was sequenced and compared with 117 publically available genomes. This strain is phylogenetically distinct and lacks virulence genes encoding the toxin, BigA and Sdr-like adhesins. Strain 5015 possesses spaD-type and spaH-type pilus gene clusters with a loss of some gene functions, and 31 unique genes that need molecular characterization to understand their potential role in virulence characteristics.

Genomic analyses reveal two distinct lineages of Corynebacterium ulcerans strains.

Corynebacteriumulcerans is an important zoonotic pathogen which is causing diphtheria-like disease in humans globally. In this study, the genomes of three recently isolated C. ulcerans strains, 4940, 2590 and BR-AD 2649, respectively from an asymptomatic carrier, a patient with pharyngitis and a canine host, were sequenced to investigate their virulence potential. A comparative analysis was performed including the published genome sequences of 16 other C. ulcerans isolates. C. ulcerans strains belong to two lineages; 13 strains are grouped together in lineage 1, and six strains comprise lineage 2. Consistent with the zoonotic nature of C. ulcerans infections, isolates from both the human and canine hosts clustered in both the lineages. Most of the strains possessed spaDEF and spaBC gene clusters along with the virulence genes cpp, pld, cwlH, nanH, rpfI, tspA and vsp1. The gene encoding Shiga-like toxin was only present in one strain, and 11 strains carried the tox gene encoding the diphtheria-like toxin. However, none of strains 4940, 2590 and BR-AD 2649 carried any toxin genes. These strains varied in the number of prophages in their genomes, which suggests that they play an important role in introducing diversity in C. ulcerans. The pan-genomic analyses revealed a variation in the number of membrane-associated and secreted proteins that may contribute to the variation in pathogenicity among different strains.

Biofilm formation on different pH conditions by Streptococcus agalactiae isolated from bovine mastitic milk.

Streptococcus agalactiae is among the most relevant aetiologic agent of bovine clinical and subclinical mastitis, a major problem for the dairy industry. In Brazil, clonal diversity, capsular typing and multidrug resistance profiles of S. agalactiae related to human and bovine infections need further investigation. Presently, S. agalactiae isolates of bovine subclinical mastitis, from Brazilian Northeastern region, were submitted to capsular and pulsed-field gel electrophoresis (PFGE)-typing, antimicrobial susceptibility and assays of biofilm formation at different time incubation and pH levels. Sixteen bovine isolates were characterized by polymerase chain reaction assay as S. agalactiae capsular type II (CTII) and classified by PFGE in A1/A2 (n = 06), B1/B2 (n = 06), C (n = 03) and D (n = 01) patterns. Bovine S. agalactiae CTII strains were classified as 25% multidrug-resistant (MDR) with susceptibility to penicillin, linezolid and vancomycin. Biofilm formation on abiotic surface was strain- and time-dependent with significantly higher rates at pH 6·5. In conclusion, S. agalactiae capsular type II isolates recovered from bovine subclinical mastitis produced different pH-dependent biofilm levels. Our findings suggest that biofilm production is modulated by environmental factors and provides S. agalactiae advantageous in colonizing mammary gland during mastitis development, including MDR strains. SIGNIFICANCE AND IMPACT OF THE STUDY: Streptococcus agalactiae is among the most relevant aetiologic agent of bovine clinical and subclinical mastitis, a major problem for the dairy industry. The disease may cause significant economic loss due to decreased production and milk quality and increased use of medicaments. Presently, data demonstrated that biofilm formation favours the establishment of infectious process in health mammary tissue by S. agalactiae and emphasizes that an acidic pH promotes adhesion by biofilm-forming bacterial strains. S. agalactiae strains (25%) showed resistance to tetracycline, azithromycin, erythromycin and clindamycin, and consequently were classified as multidrug-resistant strains.

Novel bovine-associated pVAPN plasmid type in Rhodococcus equi identified from lymph nodes of slaughtered cattle and lungs of people living with HIV/AIDS.

Rhodococcus equi is a well-recognized Gram-positive intracellular facultative bacterium that is opportunistic in nature, which causes pyogranulomatous infections in humans and multiple host animals. The pathogenicity of the microorganism has been attributed to the presence of plasmid-encoded virulence-associated proteins (Vap). To date, three host-associated virulence plasmid types of R. equi have been identified as follows: the circular pVAPA and pVAPB, related, respectively, to equine and porcine isolates, and a recently described linear pVAPN plasmid associated with bovine strains, although these three types are found in human isolates. Recent phylogenomic studies support the evidence that human R. equi infection is zoonotically acquired. Nevertheless, data regarding distribution and prevalence of the host-adapted virulence plasmid types of R. equi isolated from meat animals are scarce or unnoticed. Here, the three host-associated virulence plasmid types (pVAPA, pVAPB, and pVAPN) were investigated in 154 R. equi isolates recovered from lymph nodes of cattle with lymphadenitis (n = 31), faeces of cattle without enteric signs (n = 49), as well as different clinical specimens from human patients (n = 74). The analysis of virulence profile of 74 R. equi from humans revealed six (8.1%) isolates pVAPB (type 8), two (2.7%) pVAPN, and one (1.3%) pVAPB (type 11), all of which were from lung samples from people living with HIV/AIDS. From the lymph node samples of cattle, 41.9% (13 of 31) isolates revealed pVAPN type, whereas all isolates from faecal samples were negative for three host-associated types. Here, recently described bovine-associated pVAPN type was detected in R. equi isolates recovered from the lungs of people living with HIV/AIDS and lymph nodes from slaughtered cattle intended for human consumption; a finding that represents a public health concern, mainly in countries where undercooked or raw meat are traditionally consumed.

Streptococcus agalactiae infection in cancer patients: a five-year study.

Although the highest burden of Streptococcus agalactiae infections has been reported in industrialized countries, studies on the characterization and epidemiology are still limited in developing countries and implementation of control strategies remains undefined. The aim of this retrospective study was to assess the epidemiological, clinical, and microbiological aspects of S. agalactiae infections in cancer patients treated at a Reference Brazilian National Cancer Institute - INCA, Rio de Janeiro, Brazil. We reviewed the clinical and laboratory records of all cancer patients identified as having invasive S. agalactiae disease during 2010-2014. The isolates were identified by biochemical analysis and tested for antimicrobial susceptibility. A total of 263 strains of S. agalactiae were isolated from cancer patients who had been clinically and microbiologically classified as infected. S. agalactiae infections were mostly detected among adults with solid tumors (94 %) and/or patients who have used indwelling medical devices (77.2 %) or submitted to surgical procedures (71.5 %). Mortality rates (in-hospital mortality during 30 days after the identification of S. agalactiae) related to invasive S. agalactiae infections (n = 28; 31.1 %) for the specific category of neoplasic diseases were: gastrointestinal (46 %), head and neck (25 %), lung (11 %), hematologic (11 %), gynecologic (4 %), and genitourinary (3 %). We also found an increase in S. agalactiae resistance to erythromycin and clindamycin and the emergence of penicillin-less susceptible isolates. A remarkable number of cases of invasive infections due to S. agalactiae strains was identified, mostly in adult patients. Our findings reinforce the need for S. agalactiae control measures in Brazil, including cancer patients.

Diphtheria outbreak in Maranhão, Brazil: microbiological, clinical and epidemiological aspects.

We describe microbiological, clinical and epidemiological aspects of a diphtheria outbreak that occurred in Maranhão, Brazil. The majority of the 27 confirmed cases occurred in partially (n = 16) or completely (n = 10) immunized children (n = 26). Clinical signs and characteristic symptoms of diphtheria such as cervical lymphadenopathy and pseudomembrane formation were absent in 48% and 7% of the cases, respectively. Complications such as paralysis of lower limbs were observed. Three cases resulted in death, two of them in completely immunized children. Microbiological analysis identified the isolates as Corynebacterium diphtheriae biovar intermedius with a predominant PFGE type. Most of them were toxigenic and some showed a decrease in penicillin G susceptibility. In conclusion, diphtheria remains endemic in Brazil. Health professionals need to be aware of the possibility of atypical cases of C. diphtheriae infection, including pharyngitis without pseudomembrane formation.

Infective endocarditis in Rio de Janeiro, Brazil: a 5-year experience at two teaching hospitals.

INTRODUCTION: Despite the recent advances in diagnosis and treatment, mortality rates due to infective endocarditis (IE) remain high if not aggressively treated with antibiotics, whether or not associated with surgery. Data on the prevalence, epidemiology and etiology of IE from developing countries remain scarce. The aim of this observational, prospective cohort study was to report a 5-year experience of IE at two teaching hospitals in Rio de Janeiro, Brazil. MATERIAL AND METHODS: Demographical, anamnestic and microbiological characteristics of 71 IE patients were evaluated during the period of January 2009 to March 2013. RESULTS: The mean age of the IE patients was 49.8 ± 2.4 years, of which 41 (57.7%) were males. The median time between the onset of symptoms and diagnosis of IE was 35.8 ± 4.8 days. A total of 31 (43.6%) cases of community-acquired infective endocarditis (CAIE) and 40 (56.3%) cases of healthcare-acquired infective endocarditis (HAIE) were observed. Staphylococcus aureus (30%) was the predominant cause of IE. Streptococcus spp. (45.1 %) was the predominant cause of the CAIE while S. aureus (32.5%) and Enterococcus spp. (27.2 %) were the main etiological agents of HAIE. For 64 (90.1 %) patients with native valve endocarditis, the mitral valve was the most commonly affected (48.3%). The main source of IE in this cohort was intravascular catheter. The tricuspid valve and renal chronic insufficiency were more frequent in patients with HAIE than CAIE (p = 0.001). The risk factors associated with in-hospital mortality rate (46.4%) in IE patients were: age over 45 (OR 3.4; 95% CI 1.03-11.24; p = 0.04) and chronic renal insufficiency (OR 38.3; 95% CI 3.2-449.4; p = 0.004). CONCLUSIONS: At two main teaching hospitals in Brazil, Streptococcus spp. was the principal pathogen of CAIE while S. aureus and Enterococcus spp. were the most frequent causes of HAIE. IE remains a serious disease associated with high in-hospital mortality rate (46.6%); especially, in individuals over 45 years of age and with renal failure. Data suggest that early surgery may improve the outcome of IE patients.

A case report and literature overview: Abiotrophia defectiva aortic valve endocarditis in developing countries.

A fatal case of aortic valve endocarditis due to Abiotrophia defectiva was reported in Brazil. An overview of cases of endocarditis and other human infections related to A. defectiva in developing countries was also accomplished.

SubMICs of penicillin and erythromycin enhance biofilm formation and hydrophobicity of Corynebacterium diphtheriae strains.

Subinhibitory concentrations (subMICs) of antibiotics may alter bacterial surface properties and change microbial physiology. This study aimed to investigate the effect of a subMIC (⅛ MIC) of penicillin (PEN) and erythromycin (ERY) on bacterial morphology, haemagglutinating activity, cell-surface hydrophobicity (CSH) and biofilm formation on glass and polystyrene surfaces, as well as the distribution of cell-surface acidic anionic residues of Corynebacterium diphtheriae strains (HC01 tox(-) strain; CDC-E8392 and 241 tox(+) strains). All micro-organisms tested were susceptible to PEN and ERY. Growth in the presence of PEN induced bacterial filamentation, whereas subMIC of ERY caused cell-size reduction of strains 241 and CDC-E8392. Adherence to human erythrocytes was reduced after growth in the presence of ERY, while CSH was increased by a subMIC of both antibiotics in bacterial adherence to n-hexadecane assays. Conversely, antibiotic inhibition of biofilm formation was not observed. All strains enhanced biofilm formation on glass after treatment with ERY, while only strain 241 increased glass adherence after cultivation in the presence of PEN. Biofilm production on polystyrene surfaces was improved by ⅛ MIC of ERY. After growth in the presence of both antimicrobial agents, strains 241 and CDC-E8392 exhibited anionic surface charges with focal distribution. In conclusion, subMICs of PEN and ERY modified bacterial surface properties and enhanced not only biofilm formation but also cell-surface hydrophobicity. Antibiotic-induced biofilm formation may contribute to the inconsistent success of antimicrobial therapy for C. diphtheriae infections.

Multilocus sequence types of invasive Corynebacterium diphtheriae isolated in the Rio de Janeiro urban area, Brazil.

Invasive infections caused by Corynebacterium diphtheriae in vaccinated and non-vaccinated individuals have been reported increasingly. In this study we used multilocus sequence typing (MLST) to study genetic relationships between six invasive strains of this bacterium isolated solely in the urban area of Rio de Janeiro, Brazil, during a 10-year period. Of note, all the strains rendered negative results in PCR reactions for the tox gene, and four strains presented an atypical sucrose-fermenting ability. Five strains represented new sequence types. MLST results did not support the hypothesis that invasive (sucrose-positive) strains of C. diphtheriae are part of a single clonal complex. Instead, one of the main findings of the study was that such strains can be normally found in clonal complexes with strains related to non-invasive disease. Comparative analyses with C. diphtheriae isolated in different countries provided further information on the geographical circulation of some sequence types.

Plasticity of Corynebacterium diphtheriae pathogenicity islands revealed by PCR.

Despite the existence of a vaccine against diphtheria, this disease remains endemic and is reemerging in several regions due to many factors, including variations in genes coding for virulence factors. One common feature of virulence factors is their high concentration in pathogenicity islands (PAIs), very unstable regions acquired via horizontal gene transfer, which has lead to the emergence of various bacterial pathogens. The 13 putative PAIs in Corynebacterium diphtheriae NCTC 13129 and the reemergence of this disease point to the great variability in the PAIs of this species, which may reflect on bacterial life style and physiological versatility. We investigated the relationships between the large number of PAIs in C. diphtheriae and the possible implications of their plasticity in virulence. The GenoFrag software was used to design primers to analyze the genome plasticity of two pathogenicity islands of the reference strain (PiCds 3 and 8) in 11 different strains. We found that PiCd 3 was absent in only two strains, showing genes playing putative important roles in virulence and that only one strain harbored PiCd 8, due to its location in a putative "hotspot" for horizontal gene transfer events.

Strain-dependent arthritogenic potential of the zoonotic pathogen Corynebacterium ulcerans.

During the last decade the majority of diphtheria cases in Europe had Corynebacterium ulcerans as the etiologic agent with dogs and cats as the reservoir hosts. However, little has been documented about the virulence factors of this zoonotic pathogen. To set up an in vivo experimental C. ulcerans infection model, conventional Swiss Webster mice were intravenously infected with different doses (from 1 × 10(7) to 5 × 10(9) bacteria per mouse) of C. ulcerans strains, namely 809 (from human lower respiratory tract), BR-AD22 (from asymptomatic dog nares) and CDC-KC279. Mortality rates were demonstrated by LD(50) values ranging from 1.9 × 10(8) to 1.3 × 10(9). Viable bacteria were recovered from blood, kidneys, liver, spleen and joints. For CDC-KC279 and 809 strains (2 × 10(8)mL(-1)) approximately 85% and 72% of animals with articular lesions were observed, respectively; BR-AD22-infected mice showed no signs of arthritis. CDC-KC279 and 809 strains exhibited higher arthritogenic potential when compared to the homologous toxigenic (ATCC27012) and non-toxigenic (ATCC27010) strains of Corynebacterium diphtheriae. A high number of affected joints and arthritis index in addition to the histopathological features, including subcutaneous edema, inflammatory infiltrate, damage to bone tissue and synoviocyte hypertrophy, indicated a strain-dependent ability of C. ulcerans strains to cause severe polyarthritis. A correlation between the arthritis index and systemic levels of IL-6 and TNF-α was observed for C. ulcerans strains, with the exception of the non-arthritogenic BR-AD22 strain. In conclusion, C. ulcerans revealed a strain-dependent arthritogenic potential independent of DNAse, PLD and diphtheria toxin production.

Concurrent diphtheria and infectious mononucleosis: difficulties for management, investigation and control of diphtheria in developing countries.

We report a case of concurrent diphtheria and infectious mononucleosis in an 11-year-old Brazilian child. Two days after specific treatment for diphtheria was started the patient was discharged following clinical recovery. This case highlights the difficulties in the clinical diagnosis of diphtheria in partially immunized individuals, and for the management and control of diphtheria in developing countries.

Similarity of rpoB gene sequences of sucrose-fermenting and non-fermenting Corynebacterium diphtheriae strains.

During the last decades, the majority of Brazilian Corynebacterium diphtheriae isolates were shown to be capable to metabolize sucrose, sometimes leading to erroneous identification as a non-diphtheric Corynebacterium species. The sequencing of the polymorphic region of the RNA polymerase beta subunit-encoding gene (rpoB) is an important taxonomic tool for identification of corynebacteria. The present study aimed to investigate the rpoB gene polymorphic features of sucrose-fermenting and non sucrose-fermenting strains. The results showed that sucrose-fermenting strains presented rpoB gene polymorphic regions with more than 98% similarity with the sequences deposited in the gene bank corresponding to non sucrose-fermenting strains. Data indicate that sucrose-fermenting isolates may act as a variant of C. diphtheriae biotype mitis. In addition we alert that sucrose-fermenting strains should not be discarded as contaminants mainly in countries where the possibility of isolation of this variant is higher.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin.

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15 cells using the expression vector pQE-30. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Expression and purification of the immunogenically active fragment B of the Park Williams 8 Corynebacterium diphtheriae strain toxin

The construction of a hexahistidine-tagged version of the B fragment of diphtheria toxin (DTB) represents an important step in the study of the biological properties of DTB because it will permit the production of pure recombinant DTB (rDTB) in less time and with higher yields than currently available. In the present study, the genomic DNA of the Corynebacterium diphtheriae Park Williams 8 (PW8) vaccine strain was used as a template for PCR amplification of the dtb gene. After amplification, the dtb gene was cloned and expressed in competent Escherichia coli M15™ cells using the expression vector pQE-30™. The lysate obtained from transformed E. coli cells containing the rDTB PW8 was clarified by centrifugation and purified by affinity chromatography. The homogeneity of the purified rDTB PW8 was confirmed by immunoblotting using mouse polyclonal anti-diphtheria toxoid antibodies and the immune response induced in animals with rDTB PW8 was evaluated by ELISA and dermonecrotic neutralization assays. The main result of the present study was an alternative and accessible method for the expression and purification of immunogenically reactive rDTB PW8 using commercially available systems. Data also provided preliminary evidence that rabbits immunized with rDTB PW8 are able to mount a neutralizing response against the challenge with toxigenic C. diphtheriae.

Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil

Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3 percent males and 30.7 percent females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63 percent) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71 percent) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50 percent of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64 percent of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.

Iron-limited condition modulates biofilm formation and interaction with human epithelial cells of enteroaggregative Escherichia coli (EAEC).

AIMS: The aim of this study was to investigate the influence of low iron availability on biofilm formation and adherence to HEp-2 cells of enteroaggregative Escherichia coli (EAEC) strains isolated from diarrhoea cases. METHODS AND RESULTS: The ability of EAEC to form biofilm on a plastic surface was evaluated quantitatively and qualitatively after 3 and 18 h of incubation of strains with or without the iron chelator 2,2-dipyridyl. When submitted to low iron conditions, prototype EAEC 042 strain showed a decrease in biofilm formation. Conversely, an increase in biofilm formation was observed for the clinical EAEC strains cultured in restricted iron condition. Moreover, the reduction of iron concentration inhibited the aggregative adherence to HEp-2 cells of all EAEC strains tested. However, all effects promoted by iron chelation were suppressed by thiourea. CONCLUSIONS: Low iron availability may modulate biofilm formation and adhesive properties of EAEC strains to HEp-2 cells. SIGNIFICANCE AND IMPACT OF THE STUDY: The data obtained in this study provide useful insights on the influence of low iron conditions possibly associated with redox stress on the pathogenesis of EAEC strains.

Diphtheria toxin IgG levels in military and civilian blood donors in Rio de Janeiro, Brazil.

Serologic data on diseases that are preventable by vaccines are necessary to evaluate the success of immunization programs and to identify susceptible subgroups. In the present study, we determined serum IgG levels against diphtheria toxin of military and civilian blood donors (N = 75; 69.3% males and 30.7% females) aged 18-64 years, from the Brazilian Army Biology Institute, Rio de Janeiro, using a commercial diphtheria kit (Diphtheria IgG ELISA; IBL, Germany). Most (63%) unprotected military donors were from the older age group of 41 to 64 years. In contrast, the majority (71%) of young military donors (18 to 30 years) were fully protected. About half of the military donors aged 31 to 40 years were protected against diphtheria. Among the civilians, about 50% of persons aged 18 to 30 years and 31 to 40 years had protective antibody levels against diphtheria as also did 64% of individuals aged 41 to 64 years. All civilians had a similar antibody response (geometric mean = 0.55 IU/mL) independent of age group. Military donors aged 18-30 years had higher IgG levels (geometric mean = 0.82 IU/mL) than military donors of 41-64 years (geometric mean = 0.51 IU/mL; P > 0.05). In conclusion, the existence of a considerable proportion of susceptible adults supports the position that reliable data on the immune status of the population should be maintained routinely and emphasizes the importance of adequate immunization during adulthood.