Capsular genotype and lipooligosaccharide locus class distribution in Campylobacter jejuni from young children with diarrhea and asymptomatic carriers in Bangladesh.

Campylobacter jejuni-related diarrheal diseases is one of the major health issues among young children (0-59 months old) in low-income countries. Monitoring of the capsular (capsule polysaccharide, CPS) types of virulent C. jejuni strains in regions where the disease is endemic is of great importance for the development of a customized capsule-based multivalent vaccine. Therefore, we aimed to determine the prevalence of CPS genotypes among C. jejuni strains isolated from young children with enteritis (n = 152) and asymptomatic carriers matched by age, sex, and residence defined as the control group (n = 215) in Bangladesh. CPS genotyping was performed using a newly established multiplex polymerase chain reaction (PCR) method and lipooligosaccharide (LOS) locus classes (A-E) were characterized using PCR as well. We identified 24 different CPS genotypes among the 367 isolates. Four prevalent capsular types, HS5/31 complex (n = 27, 18%), HS3 (n = 26, 17%), HS4A (n = 10, 7%), and HS8/17 (n = 10, 7%) covered almost 50% of the strains from enteritis patients and 43% of the isolates from controls. In combination, the CPS genotype and LOS class was not discriminative between cases and controls. Dominant capsular types previously identified in C. jejuni strains isolated from patients with Guillain-Barré syndrome in Bangladesh were rarely detected in strains isolated from the young children. A similar distribution was evident among enteritis- and control-related strains when comparison was done between CPS types and LOS classes. This is the first systematic study presenting the distribution of CPS genotypes of C. jejuni strains isolated in Bangladesh from children with diarrhea and controls, with capsular genotypes HS5/31 complex, HS3, HS4A, and HS8/17 being prevalent in both. In conclusion, systematic studies are required to develop a multivalent capsule-based vaccine for children in low-income countries.

ZikaPLAN: Zika Preparedness Latin American Network.

The ongoing Zika virus (ZIKV) outbreak in Latin America, the Caribbean, and the Pacific Islands has underlined the need for a coordinated research network across the whole region that can respond rapidly to address the current knowledge gaps in Zika and enhance research preparedness beyond Zika. The European Union under its Horizon 2020 Research and Innovation Programme awarded three research consortia to respond to this need. Here we present the ZikaPLAN (Zika Preparedness Latin American Network) consortium. ZikaPLAN combines the strengths of 25 partners in Latin America, North America, Africa, Asia, and various centers in Europe. We will conduct clinical studies to estimate the risk and further define the full spectrum and risk factors of congenital Zika virus syndrome (including neurodevelopmental milestones in the first 3 years of life), delineate neurological complications associated with ZIKV due to direct neuroinvasion and immune-mediated responses in older children and adults, and strengthen surveillance for birth defects and Guillain-Barré Syndrome. Laboratory-based research to unravel neurotropism and investigate the role of sexual transmission, determinants of severe disease, and viral fitness will underpin the clinical studies. Social messaging and engagement with affected communities, as well as development of wearable repellent technologies against Aedes mosquitoes will enhance the impact. Burden of disease studies, data-driven vector control, and vaccine modeling as well as risk assessments on geographic spread of ZIKV will form the foundation for evidence-informed policies. While addressing the research gaps around ZIKV, we will engage in capacity building in laboratory and clinical research, collaborate with existing and new networks to share knowledge, and work with international organizations to tackle regulatory and other bottlenecks and refine research priorities. In this way, we can leverage the ZIKV response toward building a long-term emerging infectious diseases response capacity in the region to address future challenges.

Campylobacter jejuni capsular genotypes are related to Guillain-Barré syndrome.

In about one in a thousand cases, a Campylobacter jejuni infection results in the severe polyneuropathy Guillain-Barré syndrome (GBS). It is established that sialylated lipo-oligosaccharides (LOS) of C. jejuni are a crucial virulence factor in GBS development. Frequent detection of C. jejuni with sialylated LOS in stools derived from patients with uncomplicated enteritis implies that additional bacterial factors should be involved. To assess whether the polysaccharide capsule is a marker for GBS, the capsular genotypes of two geographically distinct GBS-associated C. jejuni strain collections and an uncomplicated enteritis control collection were determined. Capsular genotyping of C. jejuni strains from the Netherlands revealed that three capsular genotypes, HS1/44c, HS2 and HS4c, were dominant in GBS-associated strains and capsular types HS1/44c and HS4c were significantly associated with GBS (p 0.05 and p 0.01, respectively) when compared with uncomplicated enteritis. In a GBS-associated strain collection from Bangladesh, capsular types HS23/36c, HS19 and HS41 were most prevalent and the capsular types HS19 and HS41 were associated with GBS (p 0.008 and p 0.02, respectively). Next, specific combinations of the LOS class and capsular genotypes were identified that were related to the occurrence of GBS. Multilocus sequence typing revealed restricted genetic diversity for strain populations with the capsular types HS2, HS19 and HS41. We conclude that capsular types HS1/44c, HS2, HS4c, HS19, HS23/36c and HS41 are markers for GBS. Besides a crucial role for sialylated LOS of C. jejuni in GBS pathogenesis, the identified capsules may contribute to GBS susceptibility.

Comparative population structure analysis of Campylobacter jejuni from human and poultry origin in Bangladesh.

Campylobacter jejuni is the most important cause of antecedent infections leading to Guillain-Barré syndrome (GBS) and Miller Fisher syndrome (MFS). The objective of the present study was to define the genetic diversity, population structure, and potential role of poultry in the transmission of Campylobacter to humans in Bangladesh. We determined the population structure of C. jejuni isolated from poultry (n = 66) and patients with enteritis (n = 39) or GBS (n = 10). Lipooligosaccharide (LOS) typing showed that 50/66 (76 %) C. jejuni strains isolated from poultry could be assigned to one of five LOS locus classes (A-E). The distribution of neuropathy-associated LOS locus classes A, B, and C were 30/50 (60 %) among the typable strains isolated from poultry. The LOS locus classes A, B, and C were significantly associated with GBS and enteritis-related C. jejuni strains more than for the poultry strains [(31/38 (82 %) vs. 30/50 (60 %), p < 0.05]. Multilocus sequence typing (MLST) defined 15 sequence types (STs) and six clonal complexes (CCs) among poultry isolates, including one ST-3740 not previously documented. The most commonly identified type, ST-5 (13/66), in chicken was seen only once among human isolates (1/49) (p < 0.001). Amplified fragment length polymorphism (AFLP) revealed three major clusters (A, B, and C) among C. jejuni isolated from humans and poultry. There seems to be a lack of overlap between the major human and chicken clones, which suggests that there may be additional sources for campylobacteriosis other than poultry in Bangladesh.

A novel and simple mixture as point-of-use water treatment agent to produce safe drinking water.

BACKGROUND: People in rural Bangladesh have a poor understanding of the link between use of contaminated surface water and disease. An inexpensive point-of-use water treatment agent was developed to purify surface water. METHODS: Surface water was collected from various sources in Bangladesh from February 2007 to January 2008. Microbiological and physicochemical parameters of raw and treated surface water were analysed. Water was treated with a mixture of alum potash, bleaching powder and lime, or with each agent individually. RESULTS: Raw water was contaminated with bacteria, the counts for total coliforms, faecal coliforms and faecal streptococci being 26,431, 14,548 and 240 colony-forming units (cfu) 100 ml(-1), respectively. These counts fell to 0 cfu 100 ml(-1) after treatment with the mixture. The count of artificially introduced Vibrio cholerae was also reduced to 0 cfu 100 ml(-1) after treatment. Treatment of raw water altered the pH from 6.90 to 6.87, turbidity from 21.61 to 3.55 nephelometric turbidity units (NTU), residual chlorine from 0 to 0.09 mg litre(-1), conductivity from 124.03 to 229.96 µS cm(-1), and total dissolved solids from 59.40 to 199.25 mg litre(-1). All these results of treatment were within the range recommended by the WHO as acceptable for drinking water. CONCLUSION: The mixture of alum potash, bleaching powder and lime described can be safely used to disinfect contaminated surface water to make it suitable for drinking and other household purposes in Bangladesh.

Bacterial proteases: targets for diagnostics and therapy.

Proteases are essential for the proliferation and growth of bacteria, and are also known to contribute to bacterial virulence. This makes them interesting candidates as diagnostic and therapeutic targets for infectious diseases. In this review, the authors discuss the most recent developments and potential applications for bacterial proteases in the diagnosis and treatment of bacterial infections. Current and future bacterial protease targets are described and their limitations outlined.

A novel link between Campylobacter jejuni bacteriophage defence, virulence and Guillain-Barré syndrome.

Guillain-Barré syndrome (GBS) is a post-infectious disease in which the human peripheral nervous system is affected after infection by specific pathogenic bacteria, including Campylobacter jejuni. GBS is suggested to be provoked by molecular mimicry between sialylated lipooligosaccharide (LOS) structures on the cell envelope of these bacteria and ganglioside epitopes on the human peripheral nerves, resulting in autoimmune-driven nerve destruction. Earlier, the C. jejuni sialyltransferase (Cst-II) was found to be linked to GBS and demonstrated to be involved in the biosynthesis of the ganglioside-like LOS structures. Apart from a role in pathogenicity, we report here that Cst-II-generated ganglioside-like LOS structures confer efficient bacteriophage resistance in C. jejuni. By bioinformatic analysis, it is revealed that the presence of sialyltransferases in C. jejuni and other potential GBS-related pathogens correlated significantly with the apparent degeneration of an alternative anti-virus system: type II Clusters of Regularly Interspaced Short Palindromic Repeat and associated genes (CRISPR-Cas). Molecular analysis of the C. jejuni CRISPR-Cas system confirmed the bioinformatic investigation. CRISPR degeneration and mutations in the cas genes cas2, cas1 and csn1 were found to correlate with Cst-II sialyltransferase presence (p < 0.0001). Remarkably, type II CRISPR-Cas systems are mainly found in mammalian pathogens. To study the potential involvement of this system in pathogenicity, we inactivated the type II CRISPR-Cas marker gene csn1, which effectively reduced virulence in primarily cst-II-positive C. jejuni isolates. Our findings indicate a novel link between viral defence, virulence and GBS in a pathogenic bacterium.

Siglec-7 specifically recognizes Campylobacter jejuni strains associated with oculomotor weakness in Guillain-Barré syndrome and Miller Fisher syndrome.

Due to molecular mimicry, Campylobacter jejuni lipo-oligosaccharides can induce a cross-reactive antibody response to nerve gangliosides, which leads to Guillain-Barré syndrome (GBS). Cross-reactive antibodies to ganglioside GQ1b are strongly associated with oculomotor weakness in GBS and its variant, Miller Fisher syndrome (MFS). Antigen recognition is a crucial first step in the induction of a cross-reactive antibody response, and it has been shown that GQ1b-like epitopes expressed on the surface of C. jejuni are recognized by sialic acid-binding immunoglobulin-like lectin-7 (Siglec-7). We aimed to determine the epitope specificity of C. jejuni binding to Siglec-7, and correlate the outcome to disease symptoms in GBS and MFS patients. Using a well-defined GBS/MFS-associated C. jejuni strain collection, which included three sialic acid knockout strains, we found that Siglec-7 exclusively binds to C. jejuni strains that express terminal disialylated ganglioside mimics. When serological and diagnostic patient records were correlated with the Siglec-7-binding properties, we observed an association between Siglec-7 binding and the presence of anti-GQ1b antibodies in patient serum. In addition, Siglec-7 binding was associated with oculomotor weakness in GBS and MFS patients. Lipo-oligosaccharide-specific binding of C. jejuni to Siglec-7 may be an initiating event in immune recognition and presentation, and lead to anti-GQ1b antibody production and the development of ocular weakness in GBS or MFS.

Microbiological quality of complementary foods and its association with diarrhoeal morbidity and nutritional status of Bangladeshi children.

BACKGROUND/OBJECTIVES: To examine the bacteriological quality of complementary foods (CF) and to correlate the results with diarrhoeal morbidity and nutritional status of Bangladeshi children aged 6-24 months. SUBJECTS/METHODS: A total of 212 CF samples were tested, of which 72 were collected immediately before the first time of feeding (≤ 1 h of food preparation) and 140 were collected at second/third time of feeding from 140 households located in urban and rural areas of Bangladesh. Anthropometry, food frequency data and demographic information of the children were collected. RESULTS: Of the first time feeding samples, 3% from each of urban and rural areas were found to be contaminated with faecal coliforms (FC) at ≥ 100 CFU/g. E. coli was isolated from 11% and 6% of samples, and B. cereus from 8% and 6% of samples from urban and rural areas, respectively. In contrast, 33% of the second/third time feeding samples from urban areas and 19% from rural areas were contaminated with FC at ≥ 100 CFU/g (P<0.05). E. coli was isolated from 40% and 39% of samples, and B. cereus from 33% and 26% of samples from urban and rural areas, respectively. Significantly high numbers of wasted rural children had CF with a high aerobic plate count, which was also significantly associated with diarrhoeal morbidity in children. CONCLUSIONS: Around 40% of CF samples were contaminated with E. coli, which was mainly attributable to food preparation practices. Consumption of contaminated CF appeared to be associated with a higher frequency of diarrhoea and malnutrition in children.

Molecular epidemiology of tuberculosis in rural Matlab, Bangladesh.

OBJECTIVE: To characterise and classify clinical isolates collected from tuberculosis (TB) patients in rural Bangladesh and to investigate the mode of transmission. DESIGN: An epidemiological study using a combination of conventional and molecular methods was performed in a rural population of Bangladesh. A total of 168 clinical isolates were collected from TB patients. Deletion analysis, used for rapid differentiation of members of the Mycobacterium tuberculosis complex, spoligotyping and variable number tandem repeats of mycobacterial interspersed repetitive units (VNTR-MIRU) typing were used. RESULTS: Deletion analysis identified all isolates as M. tuberculosis and further divided them into 109 strains (65%) carrying the M. tuberculosis deletion region 1 (TbD1-intact or 'ancestral' strains) and 59 strains (35%) lacking this region (TbD1 or 'modern' strains). MIRU analyses showed that 149 strains (89%) had unique patterns, whereas 19 strains (11%) clustered into eight groups. The largest cluster comprised five TbD1 strains of the Beijing type. The rate of recent transmission was estimated to be 6.5%. CONCLUSIONS: Our results suggest that TB in rural Bangladesh is caused primarily by reactivation of latent infections involving TbD1 intact strains, overlaid with the recent emergence of Beijing strain clusters that include multidrug-resistant isolates.

Emergence of multidrug-resistant NDM-1-producing Gram-negative bacteria in Bangladesh.

The main objective of this study was to investigate the prevalence of bla (NDM-1) in Gram-negative bacteria in Bangladesh. In October 2010 at the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) laboratories, 1,816 consecutive clinical samples were tested for imipenem-resistant Gram-negative organisms. Imipenem-resistant isolates were tested for the bla (NDM-1) gene. Among 403 isolates, 14 (3.5 %) were positive for bla (NDM-1), and the predominant species were Klebsiella pneumoniae, Acinetobacter baumannii, and Escherichia coli. All bla (NDM-1)-positive isolates were resistant to multiple antibiotics. Among ß-lactamase genes, bla (CTX-M-1-group) was detected in ten isolates (eight bla (CTX-M-15)), bla (OXA-1-group) in six, bla (TEM) in nine, bla (SHV) in seven, and bla (VIM) and bla (CMY) in two isolates each. The 16S rRNA methylase gene, armA, was detected in five K. pneumoniae isolates and in one E. coli isolate. rmtB and rmtC were detected in a Citrobacter freundii and two K. pneumoniae isolates, respectively. qnr genes were detected in two K. pneumoniae isolates (one qnrB and one qnrS) and in an E. coli isolate (qnrA). Transferable plasmids (60-100 MDa) carrying bla (NDM-1) were detected in 7 of the 11 plasmid-containing isolates. Pulsed-field gel electrophoresis (PFGE) analysis grouped K. pneumoniae isolates into three clusters, while E. coli isolates differed significantly from each other. This study reports that approximately 3.5 % of Gram-negative clinical isolates in Bangladesh are NDM-1-producing.

Bacterial aetiology of diarrhoeal diseases and antimicrobial resistance in Dhaka, Bangladesh, 2005-2008.

Infectious diarrhoea caused by bacterial pathogens contributes to the high level of mortality in developing countries like Bangladesh. Following standard bacteriological procedures, a total of 14 428 bacterial pathogens were isolated from 56 132 stool samples and rectal swabs collected from diarrhoeal patients between 2005 and 2008. The rate of isolation and antimicrobial susceptibility data were retrospectively analysed for these isolates and among them Vibrio spp. (42·9%) were the most predominant, followed by Shigella spp. (20·3%), Aeromonas spp. (12·8%) and Salmonella spp. (6·4%). A decreasing trend in isolation of Vibrio spp. (P<0·001) and Salmonella spp. (P<0·001) was observed. While Vibrio cholerae isolates remained susceptible to ciprofloxacin, an increase in resistance was observed in Campylobacter spp. and Shigella flexneri. Variations in susceptibility to other tested antibiotics were observed among the isolated pathogens. Access to this current data will help in understanding the local burden of diarrhoeal disease and contribute to better design of prevention programmes.

Genetic characterization of Vibrio cholerae O1 strains isolated in Zambia during 1996-2004 possessing the unique VSP-II region of El Tor variant.

New variants of Vibrio cholerae O1 have appeared in different time-frames in various endemic regions, especially in Asia and Africa. Sixty-nine strains of V. cholerae O1 isolated in Zambia between 1996 and 2004 were investigated by various genotypic techniques to determine the lineage of virulence signatures and clonality. All strains were positive for Vibrio seventh pandemic Islands (VSP)-I and VSP-II and repeat toxin (RTX) gene clusters attesting their El Tor lineage. Interestingly, strains isolated in recent times (2003-2004) were identified as an altered variant (El Tor biotype that harbours El Tor type rstR but produce classical ctxB) that replaced completely the progenitor El Tor strains prevalent in 1996-1997. Recent altered variant strains differed from prototype El Tor strains isolated earlier in that these strains lacked two ORFs, VC0493 and VC0498, in the VSP-II region. PFGE analysis revealed two major clonal lineages in the strains; cluster A represented the strains isolated before 2003 and cluster B the altered strains isolated in 2003-2004. Cluster A was closely related to prototype El Tor reference strain isolated in Bangladesh in 1971. Cluster B was found to be matched with Bangladeshi altered strains but was different from the hybrid strains isolated from Mozambique and Bangladesh. This report provides important information on the genesis of altered strains of V. cholerae O1 isolated in Zambia and emphasizes the need for further studies to follow the trends of evolutionary changes.

Campylobacter bacteremia: a rare and under-reported event?

Bacteria belonging to the species Campylobacter are the most common cause of bacterial diarrhoea in humans. The clinical phenotype associated with Campylobacter infections ranges from asymptomatic conditions to severe colitis and bacteremia. In susceptible patients, Campylobacter infections are associated with significant morbidity and mortality, with both host factors and bacterial factors being involved in the pathogenesis of bacteremia. In the host, age, gender and immune-compromising conditions may predispose for Campylobacter infections, whilst the most important bacterial determinants mentioned in the literature are cytotoxin production and flagellar motility. The role of sialylated lipo-oligosaccharide (LOS) and serum resistance in bacteremia is inconclusive at this time, and the clinical significance of Campylobacter bacteremia is not yet fully understood. More emphasis on the detection of Campylobacter species from blood cultures in susceptible patients at risk for Campylobacter infections will increase our understanding of the pathogenesis and the relevance of Campylobacter bacteremia.

Purification of household water using a novel mixture reduces diarrhoeal disease in Matlab, Bangladesh.

In Bangladesh, one of the main causes of waterborne diseases is related to the use of contaminated surface water. This pilot study was conducted to determine the acceptability and effectiveness of a recently developed surface water purifying mixture to prevent diarrhoeal diseases in a rural community in Bangladesh. The mixture, using a combination of alum potash, bleaching powder and lime, is added to 15 l of surface water and mixed; the water becomes suitable for drinking after 30 min. A total of 420 households from 15 villages were provided with the mixture and were taught how to use it. Episodes of diarrhoeal disease from study families were determined from hospital records of the International Centre for Diarrhoeal Disease Research, Bangladesh (ICDDR,B) in Matlab and were compared with diarrhoea episodes among 1613 control families who were not provided with the mixture. A total of 83 diarrhoeal patients were treated at Matlab Hospital from 1613 control families, but only one patient was treated for diarrhoea from among the intervention families. Among the intervention families, 73 families decided to shift from using tube well water to surface water using the mixture. The mixture could be used as a cheaper, easier and simpler point-of-use water treatment strategy in Bangladesh.

Axonal variant of Guillain-Barre syndrome associated with Campylobacter infection in Bangladesh.

BACKGROUND: Campylobacter jejuni enteritis is the predominant bacterial infection preceding Guillain-Barré syndrome (GBS), an acute postinfectious immune-mediated polyradiculoneuropathy. The purpose of this study was to define the clinical phenotype of GBS and the relation with preceding C jejuni infections in Bangladesh. METHODS: We performed a prospective matched case-control hospital surveillance including 100 patients fulfilling the National Institute of Neurological Disorders and Stroke criteria for GBS from 2006 to 2007 in the Dhaka area of Bangladesh. Detailed clinical, electrophysiologic, serologic, and microbiologic data were obtained with a follow-up of 6 months. RESULTS: GBS affected predominantly young adult males living in rural areas. Sixty-nine percent of the patients had clinical evidence of a preceding infection. The most frequent symptom was diarrhea (36%). The majority of patients had a pure motor variant of GBS (92%) with relatively infrequent cranial nerve involvement (30%). Twenty-five percent of patients required respiratory support. Electrophysiologic studies showed that 67% of patients had an axonal variant of GBS. Eleven patients (14%) died, and 23 (29%) remained severely disabled during the follow-up. Positive C jejuni serology was found in an unprecedented high frequency of 57% as compared with 8% in family controls and 3% in control patients with other neurologic diseases (p < 0.001). C jejuni infection was significantly associated with serum antibodies to the gangliosides GM1 and GD1a, axonal neuropathy, and greater disability. CONCLUSIONS: We report an unusually high frequency of the axonal variant of Guillain-Barré syndrome in Bangladesh, associated with preceding Campylobacter jejuni infection, severe residual disability, and high mortality.

Effects of local climate variability on transmission dynamics of cholera in Matlab, Bangladesh.

Cholera is considered as a model for climate-related infectious diseases. In Bangladesh, cholera epidemics occur during summer and winter seasons, but it is not known how climate variability influences the seasonality of cholera. Therefore, the variability pattern of cholera events was studied in relation to the variation in local climate variables in Matlab, Bangladesh. Classification and regression tree (CART) and principal component analysis (PCA) were used to study the dependency and variability pattern of monthly total cholera cases. An average temperature <23.25 degrees C corresponded to the lowest average cholera occurrence (23 cases/month). At a temperature of >or=23.25 degrees C and sunshine <4.13h/day, the cholera occurrence was 39 cases/month. With increased sunshine (>or=4.13h/day) and temperature (23.25-28.66 degrees C), the second highest cholera occurrence (44 cases/month) was observed. When the sunshine was >or=4.13h/day and the temperature was >28.66 degrees C, the highest cholera occurrence (54 cases/month) was observed. These results demonstrate that in summer and winter seasons in Bangladesh, temperature and sunshine hours compensate each other for higher cholera incidence. The synergistic effect of temperature and sunshine hours provided the highest number of cholera cases.

Evidence of intra-familial transmission of Helicobacter pylori by PCR-based RAPD fingerprinting in Bangladesh.

Helicobacter pylori is a genetically diverse bacterial species, which has facilitated adaptation to new hosts and persists worldwide. The main objective of this study was to explore intra-familial transmission of H. pylori in Bangladesh. We characterized H. pylori in 35 families including 138 family members using random amplified polymorphic DNA (RAPD) fingerprinting. Forty-six percent of H. pylori isolated from the mother shared a related genotype with strains isolated from their children. Twenty-nine percent of H. pylori isolates of the mother are related to the youngest children. Only 6% of the parents shared related genotype of H. pylori. These findings suggest that mother-to-child transmission occurs in early childhood and is the most probable route of transmission of H. pylori in Bangladesh.

Rapid identification of mycobacteria by Raman spectroscopy.

A number of rapid identification methods have been developed to improve the accuracy for diagnosis of tuberculosis and to speed up the presumptive identification of Mycobacterium species. Most of these methods have been validated for a limited group of microorganisms only. Here, Raman spectroscopy was compared to 16S rRNA sequencing for the identification of Mycobacterium tuberculosis complex strains and the most frequently found strains of nontuberculous mycobacteria (NTM). A total of 63 strains, belonging to eight distinct species, were analyzed. The sensitivity of Raman spectroscopy for the identification of Mycobacterium species was 95.2%. All M. tuberculosis strains were correctly identified (7 of 7; 100%), as were 54 of 57 NTM strains (94%). The differentiation between M. tuberculosis and NTM was invariably correct for all strains. Moreover, the reproducibility of Raman spectroscopy was evaluated for killed mycobacteria (by heat and formalin) versus viable mycobacteria. The spectra of the heat-inactivated bacteria showed minimal differences compared to the spectra of viable mycobacteria. Therefore, the identification of mycobacteria appears possible without biosafety level 3 precautions. Raman spectroscopy provides a novel answer to the need for rapid species identification of cultured mycobacteria in a clinical diagnostic setting.

Molecular typing of a suspected cluster of Nocardia farcinica infections by use of randomly amplified polymorphic DNA, pulsed-field gel electrophoresis, and amplified fragment length polymorphism analyses.

Randomly amplified polymorphic DNA (RAPD), pulsed-field gelelectrophoresis (PFGE), and amplified fragment length polymorphism (AFLP) analyses were used to investigate a possible outbreak of Nocardia farcinica. RAPD and PFGE analyses yielded irreproducible and unsatisfactory results, respectively. AFLP analysis seem to be a promising and welcome addition for molecular analysis of Nocardia isolates.