Characterization of the virulence of three novel clade 2 Clostridioides (Clostridium) difficile strains and a two-year screening in animals and humans in Brazil.

Clostridioides (Clostridium) difficile infection (CDI) is an evolving global healthcare problem, and owing to the diverse and dynamic molecular epidemiology of C. difficile, new strains continue to emerge. In Brazil, only two cases of CDI due to the so called hypervirulent PCR ribotype (RT) 027 belonging to clade 2 have ever been reported, whereas incidence of CDI due to another "hypervirulent" RT078 (clade 5) has not yet been reported. In contrast, novel clade 2 strains have been identified in different hospitals. To better understand the epidemiology of CDIs in Brazil, this study aimed to genotypically and phenotypically characterize three novel Brazilian clade 2 strains (RT883, 884, and 885) isolated from patients with confirmed CDI. In addition, to better understand the circulating RTs, a two-year sampling was conducted in patients from the same hospital and in several domestic and wild animal species. The three strains examined showed lower production of A/B toxins than the control RT027, although two of these strains harbored a truncated tcdC gene. All strains showed swimming motility similar to that of RT027, while RT883 showed higher spore production than the reference strain. In the in vivo hamster model, the lethality of all strains was found to be similar to that of RT027. Both cgMLST and cgMLSA analyses revealed a high genetic similarity among the three-novel clade 2 isolates. In the two-year survey in animals and humans, RT883, 884, and 885 were not detected; however, three new RTs (RT988, RT989, and RT990) were isolated, two of which were genetically related to the three previously reported clade 2 strains. RT106 and RT126 were most frequently detected in humans (47.9%) and animals (57.9%), respectively. Furthermore, RT027 and RT078 were not detected in humans. The results of this study suggest that these novel clade 2 strains have virulence potential and that new strains from clade 2 continue to emerge in our setting, indicating the need for long-term local surveillance.

Emerging infectious colitis.

PURPOSE OF REVIEW: The present review will highlight recent advances in the knowledge of emerging pathogens causing infectious colitis and provide a description of the most important food-borne outbreaks. RECENT FINDINGS: Outbreaks of enteric disease caused by Salmonella spp., Campylobacter spp., and Shiga toxin-producing Escherichia coli (STEC) continue to surprise with new epidemiological findings or changing virulence characteristics. These pathogens evolve to exploit novel opportunities for spread and transmission, such as fresh produce within the food chain, and generate new public health challenges. Organic sprouts were recently considered as the source responsible for a large German disease outbreak comprising 3842 patients. The outbreak strain was identified as an enteroaggregative STEC O104:H4 (EAggC), a rare hybrid pathogen that harbours the phage encoded Shiga toxin gene and antibiotic resistance in an EAggEC background. Clostridium difficile PCR ribotype 078 is emerging across Europe, causing severe disease outside healthcare facilities as well as disease in farm animals, indicating that the species border has been crossed. Although the global impact of cryptosporidiosis is less pronounced, these organisms have been responsible for large outbreaks of infectious diarrhoea, often not reported. Invasive listeriosis is a serious food-borne illness and was found recently in 28 US states affecting 147 patients, associated with eating contaminated cantaloupe. Outbreaks of gastroenteritis caused by Listeria monocytogenes are most likely severely underestimated. Centralized surveillance of food-borne enteropathogens is essential for the early detection of disease outbreaks and for the organization of an immediate and appropriate response. SUMMARY: An improved understanding of the pathogenesis, pathology and epidemiology of emerging enteropathogens causing infectious colitis will provide new approaches for disease prevention and control.

Intravenous tigecycline as adjunctive or alternative therapy for severe refractory Clostridium difficile infection.

Clostridium difficile infection (CDI) has become more refractory to standard therapy. We describe 4 patients with severe refractory CDI who were successfully treated with tigecycline. Symptoms improved within 1 week. No relapses were observed. This favorable outcome suggests that tigecycline might be a useful alternative for treating severe refractory CDI.

Application of real-time PCR to recognize atypical mycobacteria in archival skin biopsies: high prevalence of Mycobacterium haemophilum.

Atypical mycobacterial skin infections are difficult to diagnose owing to their aspecific histopathologic presentations and to the presence of few bacteria. Therefore, these infections are often not recognized. Molecular detection of mycobacterial DNA has proven to be useful in clinical samples. The aim of this study was to investigate the incidence of mycobacterial involvement in skin biopsies showing granulomatous inflammation, using real-time polymerase chain reaction (PCR). Real-time PCR specific for the genus Mycobacterium and the species Mycobacterium avium and Mycobacterium haemophilum was performed on formalin-fixed/paraffin-embedded biopsies from patients with granulomatous inflammation of the skin, from the period 1984 to 2004. A control group was assembled from patients with proven basal cell carcinoma. Amplicons of all positive reactions were sequenced to confirm or identify the mycobacterial species. Of 30 patients, 13 (43%) were found to be positive for mycobacterial infection, of whom only 5 patients had been previously diagnosed with a mycobacterial disease. M. haemophilum was identified as the most common species (n=7). The other identified species were Mycobacterium malmoense, Mycobacterium gordonae, and Mycobacterium marinum. The results show that real-time PCR is useful in detecting mycobacterial infections in undiagnosed formalin-fixed/paraffin-embedded skin samples and that the application of molecular approaches would improve the diagnoses of mycobacterial skin infections.

Mycobacterium haemophilum and lymphadenitis in children.

Infections associated with Mycobacterium haemophilum are underdiagnosed because specific culture methods required for its recovery are not applied routinely. Using polymerase chain reaction (PCR) technology on fine needle aspirates and biopsied specimens from 89 children with cervicofacial lymphadenitis, we assessed the importance of M. haemophilum. Application of a Mycobacterium genus-specific real-time PCR in combination with amplicon sequencing and a M. haemophilum-specific PCR resulted in the recognition of M. haemophilum as the causative agent in 16 (18%) children with cervicofacial lymphadenitis. M. avium was the most frequently found species (56%), and M. haemophilum was the second most commonly recognized pathogen. Real-time PCR results were superior to culture because only 9 (56%) of the 16 diagnosed M. haemophilum infections were positive by culture.

Long term effects of vaccination of patients deficient in a late complement component with a tetravalent meningococcal polysaccharide vaccine.

Of 45 Russian patients with late complement component deficiency (LCCD) who experienced one to five meningococcal infections, 31 were immunised with a meningococcal A/C/W135/Y polysaccharide vaccine and were followed for 3-8 years. Total and immunoglobulin (Ig) class specific concentration of antibodies to meningococcal polysaccharides in sera of LCCD patients increased significantly 1 month after vaccination and remained elevated for 3 years. Revaccination of LCCD patients 3 years after the first dose restored the total Ig concentrations to those observed 1 year after the first vaccination. Six new episodes of meningococcal infection in four patients developed in the group of 31 vaccinees; six episodes in six patients developed in the same time in the group of 14 non-vaccinated LCCD persons. Survival analysis demonstrated the risk to contract meningococcal disease decreased significantly for vaccinees in comparison to non-vaccinees (P<0.05). Vaccination with of meningococcal tetravalent polysaccharide vaccine decreases the risk of meningococcal infection in LCCD patients.

Antibody-dependent killing of meningococci by human neutrophils in serum of late complement component-deficient patients.

BACKGROUND: Thirty-one Russian patients with late complement component deficiency (LCCD) who had experienced one to five meningococcal infections were immunized with meningococcal polysaccharide vaccine (A + C + W135 + Y) and were followed for 3-8 years. We investigated the potentially protective killing effect of human neutrophils (PMNL) on serogroup A and W135 meningococci. METHODS: Meningococci were incubated in LCCD vaccine sera in the absence or presence of PMNL, and the number of live bacteria (CFU) was determined by plating onto chocolate agar. RESULTS: When meningococci were incubated in the LCCD sera alone, exponential growth of meningococci occurred despite the presence of meningococcal antibodies. After the addition of PMNL, meningococci were inhibited in their growth or even eliminated. Group A or W135 meningococci were killed effectively by PMNL in 80% of the sera which were collected 1 month to 1 year after vaccination compared to only 40% in the prevaccination LCCD sera (p < 0.05). Three years after vaccination 67% of the LCCD sera were still capable of promoting killing (and even 90% after revaccination). The rate of killing correlated with the concentration of serogroup-specific immunoglobulins. In 83% of the 72 LCCD sera with more than 5 microg/ml anti-group A immunoglobulins the killing of group A meningococci was promoted. By contrast, only 21% of 19 samples with lower specific antibody levels showed a PMNL-mediated meningococcal killing (p < 0.05). The same effect was observed for group W135 meningococci. CONCLUSION: PMNL kill meningococci during incubation in LCCD serum; this effect increases after vaccination and depends on both specific antibody and complement. Protection by vaccination may therefore be caused by an increased killing capacity of PMNL.