Conventional Microbiology Research Articles

Development of a visible loop mediated isothermal amplification assay for rapid detection of Bacillus anthracis

Distressing effects on animal and human health with lethal progression, being used as bioweapon and shared features with non-pathogenic bacteria demands sensitive, specific, safe, cost effective and rapid detection methods for anthrax causing organisms. Conventional microbiology based diagnostics for anthrax are time consuming and need sophisticated equipment, while molecular diagnostics require less time and labor. The Loop mediated isothermal amplification assay (LAMP) is rapid, sensitive and specific assay and requires no specialized equipment. In the present study, we developed a LAMP assay for rapid as well as specific detection of Bacillus anthracis. The optimized assay produced positive results with the Sterne strain and one field isolate of B. anthracis and, negative results with other bacteria of the same and different genera within 2 h. Sensitivity was 500 fg of total DNA of B. anthracis, which was 100 times more sensitive than conventional PCR. The present study also demonstrated that the simple method of total DNA extraction by repeated boiling and freezing will not adversely affect the LAMP results. In conclusion, the optimized LAMP assay is a promising tool for the specific, sensitive, less time-consuming diagnosis for anthrax causing bacteria and also, for detecting the virulence of suspected B. anthracis cultures.

Features of Streptococcus agalactiae strains recovered from pregnant women and newborns attending different hospitals in Ethiopia

BackgroundStreptococcus agalactiae (Group B Streptococcus, GBS) serotypes, sequence types, and antimicrobial resistance profile vary across different geographic locations affecting disease patterns in newborns. These differences are important considerations for vaccine development efforts and data from large countries in Africa is limited. The aim of this study was to determine serotypes and genotypes of GBS isolates from pregnant women and their newborns in Ethiopia.MethodsA hospital based cross-sectional study was conducted at three hospitals in Ethiopia from June 2014 to September 2015. Out of 225 GBS isolates, 121 GBS were recovered, confirmed and characterized at CDC’s Streptococcus Laboratory using conventional microbiology methods and whole genome sequencing.ResultsOf the 121 isolates, 87 were from rectovaginal samples of pregnant women, 32 from different body parts of their newborns and 2 from blood of newborns with suspected sepsis. There were 25 mother-infant pairs and 24 pairs had concordant strains. The most prevalent serotypes among mothers and/or their babies were II, Ia and V (41.5, 20.6, 19.5 and 40.6%, 25 and 15.6%, respectively). Multilocus sequence typing (MLST) on 83 isolates showed ST10 (24; 28.9%) and ST2 (12; 14.5%) as most predominant sequence types. All GBS strains were susceptible to penicillin, cefotaxime and vancomycin, which correlated to the presence of wildtype PBP2x types and the lack of known vancomycin-resistance genes. Tetracycline resistance was high (73; 88%, associated primarily with tetM, but also tetO and tetL). Five isolates (6%) were resistant to erythromycin and clindamycin and 3 isolates were fluoroquinolone-resistant, containing associated mutations in gyrA and parC genes. All isolates were positive for one of four homologous Alpha/Rib family determinants and 1–2 of the three main pilus types.ConclusionsPredominant serotypes were II, Ia, and V. A limited number of clonal types were identified with two STs accounting for about half of the isolates. All strains collected in this study were susceptible to beta-lactam antibiotics and vancomycin. Typical of most GBS, these isolates were positive for single alpha-like family protein, serine-rich repeat gene, as well as 1–2 pilus determinants.

Identification of Bacterial and Fungal Pathogens by rDNA Gene Barcoding in Vitreous Fluids of Endophthalmitis Patients

ABSTRACT Purpose: To identify the bacterial and fungal pathogens in ocular samples of clinically suspected endophthalmitis patients by conventional culture methods and 16S and 28S rDNA gene sequencing respectively. Methods: A total of 88 patients with clinically suspected endophthalmitis were included in this study. Under sterile operating conditions, a vitreous fluid (0.1–0.2 ml) was obtained by pars plana vitrectomy procedure. The samples were processed for conventional microbiology methods and PCR. PCR targeting 16S rDNA gene for bacteria and 28S rDNA gene for fungus were performed individually using the MightyAmp DNA Polymerase Ver. 2 (TaKaRa China) kit. The PCR amplified samples were sequenced and aligned using CLUSTAL-W tool. The phylogenetic tree was constructed by Neighborhood joining along with the reference sequences downloaded from NCBI database using MEGA X software. Results: 67 Post-operative, 12 Endogenous and 9 traumatic endophthalmitis patients were included as study subjects. By the direct culturing bacterial growth was observed in 17 samples and fungal growth in three samples. PCR was positive for all the culture positive samples, in addition 14 were positive in culture negative samples. The predominant species identified in gram-positive bacteria were Staphylococcus spp., and Pseudomonas spp. in the gram-negative group. Both PCR and culture identified only three samples positive for fungal pathogens which were identified as Aspergillus fumigatus, Candida albicans, and Exerohilum rostratum. Conclusions: PCR based molecular diagnosis is more sensitive than the conventional gold standard culture methods in endophthalmitis. Bacterial pathogens were found to be the predominant in causing endophthalmitis than fungal pathogens.

De-escalation of Antibiotics in Severe Sepsis and Septic Shock at a Large Municipal Hospital

Background: Early de-escalation of antibiotics in sepsis may be safe and effective. In our study, we performed a retrospective chart review of patients admitted to a large municipal hospital who were treated for severe sepsis or septic shock to compare outcomes in patients who experienced early de-escalation (DEG) with outcomes of those who did not (NDG).Methods: The observational study was conducted at Bellevue Hospital Center (an 850-bed municipal hospital affiliated with New York University School of Medicine, New York, NY). Patients admitted from January 1 to December 31, 2015, who were treated for severe sepsis or septic shock during any time of their hospital stay were reviewed for the study. De-escalation was defined as narrowing or discontinuation of 1 or more antimicrobial therapies < 3 days after sepsis onset. Results: Overall, 277 patients were included (DEG, 90 patients, 32%; NDG, 187 patients, 68%). The groups were similar in terms of sex, comorbidities, length of stay, and severity of illness: septic shock (47% DEG vs 49% NDG; P = .693) and ICU stay (27% DEG vs 32% NDG; P = .406). DEG patients were slightly older than NDG patients: (DEG age, 63+16 years vs NDE age, 58+16 years; P = .028). There was no difference in hospital mortality (8% DEG vs 12% NDE; P = .257). Nearly half of the patients in both groups (46% DEG and 47% NDG) had no causative microorganisms identified using conventional microbiology culture. The common sources of primary infection were respiratory, urinary tract, and gastrointestinal infections, and these were not different between groups. Also, 69% of DEG patients and 79% of NDG patients received antibiotics for >7 days (P = .002). Empiric intravenous vancomycin was initiated in 83% in DEG patients and 74% in NDG patients at sepsis diagnosis. Although organisms covered by intravenous vancomycin were isolated from only 17% of patients in DEG and 23% in NDG, vancomycin was continued for >5 days in 34% of DEG patients and 50.3% of NDG patients (P < .001). 60% of patients in DEG and 61% in NDG were seen by infectious diseases specialists (ID). Patients with infectious diseases consultations had significantly more comorbidities, were more frequently in the ICU, had higher MDRO isolation and longer hospital stays, but they were still de-escalated without a difference in mortality. Conclusions: Microbiology data did not contribute to early de-escalation of antibiotics in this study. This finding may be related to the high percentage of negative culture and unavailability of rapid molecular diagnostic tests. Shorter duration of antibiotics (including vancomycin) was not associated with worse outcome in these severely ill patients.Funding: NoneDisclosures: None

Functional selection of bacteria in an activated sludge reactor for application in saline wastewater treatment in Kerkennah, Tunisia

Domestic wastewater entering the wastewater treatment plant (WWTP) of Kerkennah (Tunisia) contains high organic load and high salinity. The biological treatment of these influents with conventional microorganisms has proven to be not very effective, which might be improved by functionally selected halotolerant/halophilic heterotrophic bacteria. Therefore, a laboratory-scaled activated sludge reactor (ASR) was used for a continuous treatment of this wastewater. The performance of the ASR, inoculated with a mixed culture of active biomass gradually acclimated to the wastewater, was evaluated in terms of carbonaceous pollutant removal efficiency. Experiments were conducted at different organic loading rates (OLRs, varying from 250 to 1200 mg chemical oxygen demand (COD)/l.d). The descriptive analysis showed that the removal efficiency (r) of the ASR was high, peaking at 95% for CODr in medium OLRs, and up to 99% for Biological oxygen demand (BODr) and total suspended solids for high OLRs and salinity. The overall removal efficiency was improved by 1/3th or more compared to the WWTP by these selected and acclimated bacteria. The different observations were combined in an innovative statistical design of Hierarchical clustering. This method improves on traditional approaches by allowing flexibility in understanding the complex variations within the WWTP and the ASR. The identification of the bacterial biomass responsible for high BODr in the ASR by conventional microbiology demonstrated the dominance of Proteus mirabilis and Aeromonas hydrophila. The 16S rRNA gene gave a more accurate identification, indicating a great bacterial diversity and the possibility of a new species.

Fluorescence In Situ Hybridization and Polymerase Chain Reaction to Detect Infections in Patients With Left Ventricular Assist Devices.

The development of driveline infections following left ventricular assist device (LVAD) implantation remains a major problem. We investigated the impact of fluorescence in situ hybridization (FISH) combined with 16S rRNA gene sequencing on the diagnosis of driveline infections. LVAD drivelines (n = 61) from 60 consecutive patients were obtained during LVAD explantation and subjected to FISH analysis. 16S rRNA gene polymerase chain reaction (PCR) and sequencing to identify the microorganisms were performed. Results were compared with those of a standard microbiological culture. The reasons for pump removal were heart transplantation (n = 22), weaning (n = 14), pump exchange due to pump thrombosis (n = 12), technical problems (n = 7), or death (n = 5). Of the 60 patients, 26 exhibited clinical signs of a VAD-specific infection, while 34 (with 35 drivelines) showed no clinical signs of infection before explantation. The spectrum of identified pathogens differed between FISH/PCR and conventional microbiological diagnostics. In general, the bacterial spectrum was more diverse in FISH/PCR as compared with conventional microbiology, which more often showed only typical skin flora (coagulase-negative staphylococci and Corynebacteriaceae). In addition to identifying the species, FISH/PCR provided information about the spatial distribution and invasiveness of the microorganisms. Cultures usually represent the only source of microbiological information for clinicians and often prove to be unsatisfactory in complex LVAD cases. FISH/PCR not only identified a greater number and variety of microorganisms than standard culture did, but it also provided information about the number, localization, and biofilm state of the pathogens, making it a useful tool for diagnosing the specific cause of LVAD driveline infections.

Phenotypically distinguishing ESBL-producing pathogens using paper-based surface enhanced Raman sensors

Antimicrobial stewardship practices are critical in preventing the further erosion of treatment options for bacterial infections. Yet, at the same time, determination of an infection’s antimicrobial susceptibility requires multiple rounds of culture and expensive lab automation systems. In this work, we report the use of paper-based surface enhanced Raman spectroscopy (SERS) sensors and portable instrumentation to phenotypically discriminate multi-drug resistance with fewer culture steps than conventional clinical microbiology. Specifically, we demonstrate the identification of resistance to varying generations of β-lactam antibiotics by detecting the activity of particular β-lactamase enzymes in a multiplexed assay. The method utilizes molecular reporters that consist of β-lactams with SERS barcodes. Hydrolysis of the β-lactam by β-lactamase enzymes in the sample expels the barcode; the released sulfur-containing barcode is then detected via SERS. Using this approach, we demonstrate the differentiation of E. coli strains with (1) extended spectrum β-lactamase (ESBL), (2) narrow-spectrum β-lactamase, and (3) no resistance, using only a single measurement on a single sample. In addition, we experimentally validate an approach to expand the library of reporters through the simple chemical synthesis of new barcoded β-lactams. Importantly, the reported method determines the susceptibility based on phenotypic β-lactamase activity, which is aligned with current microbiology lab standards. This new method will enable the precise selection of effective β-lactam antibiotics (as opposed to defaulting to drugs of last resort) faster than current methods while using simple steps and low-cost portable instrumentation.

Short communication: Characterization of an atypical maltose-negative Staphylococcus aureus through the use of phenotypic and molecular techniques

The most clinically relevant staphylococci in veterinary medicine are those that are coagulase-positive, namely Staphylococcus aureus. During microbiological udder health monitoring (2009-2018), a new S. aureus strain (coagulase-positive and maltose-negative) was discovered as an emerging udder pathogen during routine examinations of South African dairy herds. This study challenged the conventional microbiological diagnosis of staphylococci by comparing its results to those of the MALDI-TOF mass spectrometry and 16S rRNA sequencing. Both of these tests confirmed that the maltose-negative staphylococcus (MNS), identified as Staphylococcus pseudintermedius by conventional microbiology, was S. aureus ST2992. Multi locus sequence typing was performed on 3 of the MNS isolates and indicated that these isolates were of single origin. These strains tested positive for both MALA and MALR genes (control: S. aureus ATCC 25923). Although the α-glucosidase gene was present, it was not expressed phenotypically. The latter may be attributed to the abnormal stop codon identified in the MALA gene sequence of S. aureus ST2992 (GenBank accession number, MN531305). The newly identified MNS has a field behavior different to that of maltose-positive S. aureus, and more similar to the low virulence of non-aureus staphylococci.

Impact of routine vaccination against Haemophilus influenzae type b in The Gambia: 20 years after its introduction.

BackgroundIn 1997, The Gambia introduced three primary doses of Haemophilus influenzae type b (Hib) conjugate vaccine without a booster in its infant immunisation programme along with establishment of a population-based surveillance on Hib meningitis in the West Coast Region (WCR). This surveillance was stopped in 2002 with reported elimination of Hib disease. This was re-established in 2008 but stopped again in 2010. We aimed to re-establish the surveillance in WCR and to continue surveillance in Basse Health and Demographic Surveillance System (BHDSS) in the east of the country to assess any shifts in the epidemiology of Hib disease in The Gambia.MethodsIn WCR, population-based surveillance for Hib meningitis was re-established in children aged under-10 years from 24 December 2014 to 31 March 2017, using conventional microbiology and Real Time Polymerase Chain Reaction (RT-PCR). In BHDSS, population-based surveillance for Hib disease was conducted in children aged 2-59 months from 12 May 2008 to 31 December 2017 using conventional microbiology only. Hib carriage survey was carried out in pre-school and school children from July 2015 to November 2016.ResultsIn WCR, five Hib meningitis cases were detected using conventional microbiology while another 14 were detected by RT-PCR. Of the 19 cases, two (11%) were too young to be protected by vaccination while seven (37%) were unvaccinated. Using conventional microbiology, the incidence of Hib meningitis per 100 000-child-year (CY) in children aged 1-59 months was 0.7 in 2015 (95% confidence interval (CI) = 0.0-3.7) and 2.7 (95% CI = 0.7-7.0) in 2016. In BHDSS, 25 Hib cases were reported. Nine (36%) were too young to be protected by vaccination and five (20%) were under-vaccinated for age. Disease incidence peaked in 2012-2013 at 15 per 100 000 CY and fell to 5-8 per 100 000 CY over the subsequent four years. The prevalence of Hib carriage was 0.12% in WCR and 0.38% in BHDSS.ConclusionsAfter 20 years of using three primary doses of Hib vaccine without a booster Hib transmission continues in The Gambia, albeit at low rates. Improved coverage and timeliness of vaccination are of high priority for Hib disease in settings like Gambia, and there are currently no clear indications of a need for a booster dose.

Prevalence and antimicrobial susceptibility of Salmonella and Shigella species isolated from diarrheic children in Ambo town

BackgroundDiarrhea, particularly of enteric bacterial pathogen, remains a major cause of morbidity and mortality in Ethiopia. Despite the high prevalence of diarrheal disease among under-five children, antibiotic resistance of bacterial pathogens test is not part of routine childcare in the study area. This study aimed to investigate the prevalence and antimicrobial susceptibility status of Salmonella and Shigella species among diarrheic children attending public health institutions in Ambo town, west Showa, Ethiopia.MethodsInstitutional based, cross-sectional study was carried out from January to July 2014 among 239 diarrheic children below five years of age in Ambo town, Ethiopia. Information about patient demographics, signs, and symptoms was obtained from the parents/guardians of each child using a questionnaire. Stool samples from diarrheic children were collected and processed for isolation of Salmonella and Shigella using conventional microbiology procedures. Suspected Salmonella isolates were confirmed by genus-specific PCR and serotyped using a slide agglutination test. Susceptibility to 10 commonly used antimicrobials was assessed using the Kirby Bauer disc diffusion method.ResultsFrom the 239 children screened, only nine (3.8%) of them were positive for either Salmonella (n = 3) or Shigella (n = 6) and 19 (7.9%) positive for the intestinal parasite. Three species of Shigella were identified: Shigella flexinari (n = 3), Shigella boydii (n = 2), and Shigella sonnei (n = 1). The three Salmonella isolates were S. chicago, S. caracas, and S. saintpaul. Salmonella and Shigella isolates were resistant to ampicillin (88.9%), followed by tetracycline (66.7%), cotrimoxazole (55.6%), chloramphenicol (44.4%), amoxicillin (33.3%), nalidixic acid (11.1%) and cefotaxime (11.1%). All isolates were sensitive to amikacin, ciprofloxacin, and gentamycin.ConclusionIn this study, either Salmonella or Shigella species were detected only in 3.8% of diarrheic children in Ambo town, suggesting the dominance of other causes of diarrhea in the study area. A further study targeting other causes of diarrhea should be conducted to establish the major causes of childhood diarrhea in the study area.

Molecular Characterization of Bacillus-Genus Bacteria with Fibrinolytic Potential Isolated from Squashes «NTETE» in Brazzaville in the Republic of Congo

Thromboses appearing in the blood and causing cardiovascular disease by the presence of fibrin remain a problem of concern worldwide. The fibrinolytic enzymes developed by bacteria of the Bacillus-genus are today an interesting and promising alternative to chemicals with multiple consequences. It is in this perspective that three (3) samples of cooked squash wrapped in sheets and consumed in Brazzaville and collected and in three Brazzaville markets were explored. The estimated count of the total flora of each sample for each of the three markets, namely Total, Moukondo and Tsiémé was carried out on PCA and represents respectively (5.2 ± 0.12) 107 CFU / g, (1.73 ± 0 , 16) 107 CFU / g, and (9.43 ± 1.06) 107 CFU / g while bacteria of the genus Bacillus are estimated in Mossel respectively at (3.5 ± 1.16) 106 CFU / g, (4, 01 ± 0.85) 106 CFU / g (8.96 ± 0.60) 106 CFU / g. Sixty-six isolates of bacteria of the genus Bacillus isolated from squash by conventional microbiology techniques have been phenotypically characterized. The morphological types characterized are essentially the bacillary form and the spherical form. The ability to produce fibrinolytic enzymes correlated with growth was assessed. The growth in terms of optical density varies from 0.800 to 0.97 and the enzymatic production in all the isolates tested varies from 12 to 21 mm. After DNA extraction from 36 isolates, PCR amplification of the rR16S gene revealed fragments of approximately 1500bp by electrophoresis on Agarose Gel. The sequencing of thirty-four (34) fragments made it possible to obtain fifteen (15) sequences having a strong similarity between them and also with the homologs of the databases (97% to 100%) and therefore the molecular identification of: Bacillus sp 40%, Bacillus amyloliquefaciens 6.66%, Bacillus subtilis 33.33%, Bacillus Pumilus 6.66%, Bacillus megaterum 6.66%, Bacillus velezensis 6.66%. Five (5) of these sequences have been submitted to GenBank and the accession numbers are successively: MK193815.1 (Bacillus subtilis strain ASM1), MK207434.1 (Bacillus subtilis strain ASM3), MK207435.1 (Bacillus pumilus strain ASM5) , MK207436.1 (Bacillus subtilis strain ASM4), MK207437.1 (Bacillus megaterium strain ASM2).The multiple alignment of sequences obtained shows a high conservation of this gene in bacteria of the genus Bacillus. The phylogenetic classification clearly shows this monophyletic class of bacteria of the genus Bacillus with very short distances(less than 3%).

Utility of Fungal Cell Markers in Pediatric Bone Marrow Transplant Recipients for Screening and Diagnosis of Invasive Fungal Infections

Invasive fungal disease (IFD) is a significant contributor to morbidity and mortality in pediatric patients undergoing hematopoietic stem cell transplantation (HSCT). Early detection and timely initiation of antifungal therapy is important for improved outcome, but diagnosis remains difficult, especially when relying on conventional microbiology methods such as culture and microscopy. Data on the use of fungal biomarkers for screening and diagnosis of IFD in HSCT pediatric patients are limited, precluding the development of specific guidelines in this population. In this review, we have summarized available literature on kinetics, diagnostic and prognostic performance, and limitations of fungal biomarkers for screening and diagnosis of IFD in pediatric HSCT recipients to help guide their use and interpretation.

Antimicrobial and Disinfectant Susceptibility of Salmonella Serotypes Isolated from Swine Slaughterhouses.

Salmonella remains one of the most common foodborne pathogens worldwide, and its resistance to antimicrobials and disinfectants has increased considerably over the years. Thus, monitoring its resistance to products commonly used in swine production is indispensable for the development of strategies to reduce the occurrence of bacterial resistance. In this context, our aim was to detect Salmonella at different points in swine slaughterhouses, identify the main serotypes, and evaluate their resistance to disinfectants and antimicrobials used in swine production. Salmonella at the processing plants was detected by conventional microbiology. Salmonella strains were tested for susceptibility to peracetic acid (0.5% and 1%), quaternary ammonium (0.5%), and seven antimicrobials. Twenty-eight percent of the samples were positive for Salmonella, with the most identified serotypes being Salmonella Derby, Salmonella Typhimurium, and monophasic Salmonella Typhimurium. All tested strains were susceptible to both concentrations of peracetic acid, but only 28% were susceptible to quaternary ammonium. Sixteen percent of the strains were susceptible to all tested antimicrobials. Only enrofloxacin was efficient in inhibiting the growth of all strains. The highest number of non-susceptible strains was to amoxicillin, followed by chloramphenicol, florfenicol, and doxycycline. Thirty-six percent of the strains were classified as multidrug-resistant. Salmonella were detected in all slaughtering processes, and important serotypes were recovered, including Salmonella Typhimurium, Salmonella Derby, monophasic Salmonella Typhimurium, and Salmonella Infantis. We observed high rates of resistance to quaternary ammonium and to important antimicrobial agents. Salmonella Typhimurium and Salmonella Infantis were the most resistant serotypes.

Multiplex bacterial polymerase chain reaction in a cohort of patients with pleural effusion

BackgroundThe identification of the pathogens in pleural effusion has mainly relied on conventional bacterial culture or single species polymerase chain reaction (PCR), both with relatively low sensitivity. We investigated the efficacy of a commercially available multiplex bacterial PCR assay developed for pneumonia to identify the pathogens involved in pleural infection, particularly empyema.MethodsA prospective, monocentric, observational study including 194 patients with pleural effusion. Patients were evaluated based on imaging, laboratory values, pleura ultrasound and results of thoracentesis including conventional microbiology studies during hospitalisation. Multiplex bacterial PCR (Curetis Unyvero p55) was performed in batch and had no influence on therapeutic decisions.ResultsOverall, there were 51/197 cases with transudate and 146/197 with exudate. In 42% (n = 90/214) there was a clinical suspicion of parapneumonic effusion and the final clinical diagnosis of empyema was made in 29% (n = 61/214) of all cases. The most common microorganisms identified in the cases diagnosed with empyema were anaerobes [31] followed by gram-positive cocci [10] and gram-negative rods [4]. The multiplex PCR assay identified more of the pathogens on the panel than the conventional methods (23.3% (7/30) vs. 6.7% (2/30), p = 0.008).ConclusionThe multiplex PCR-based assay had a higher sensitivity and specificity than conventional microbiology when only the pathogens on the pneumonia panel were taken into account. A dedicated pleural empyema multiplex PCR panel including anaerobes would be needed to cover most common pathogens involved in pleural infection.

Appropriate Use and Future Directions of Molecular Diagnostic Testing.

Purpose of ReviewMajor technologic advances in two main areas of molecular infectious disease diagnostics have resulted in accelerated adoption or ordering, outpacing implementation, and clinical utility studies. Physicians must understand the limitations to and appropriate utilization of these technologies in order to provide cost-effective and well-informed care for their patients.Recent FindingsRapid molecular testing and, to a lesser degree, clinical metagenomics are now being routinely used in clinical practice. While these tests allow for a breadth of interrogation not possible with conventional microbiology, they pose new challenges for diagnostic and antimicrobial stewardship programs. This review will summarize the most recent literature on these two categories of technologic advances and discuss the few studies that have looked at utilization and stewardship approaches. This review also highlights the future directions for both of these technologies.SummaryThe appropriate utilization of rapid molecular testing and clinical metagenomics has not been well established. More studies are needed to assess their prospective impacts on patient management and antimicrobial stewardship efforts as the future state of infectious disease diagnostics will see continued expansion of these technologic advances.

Detection and quantification of Campylobacter spp. in Brazilian poultry processing plants.

Campylobacteriosis is considered the most common bacteria-caused human gastroenteritis in the world. Poultry is a major reservoir of Campylobacter. Human infection may occur by consumption of raw and undercooked poultry or by contamination of other foods by these items. The aim of this study was to assess the prevalence of Campylobacter spp. in poultry processing plants with conventional culture method and real-time PCR. A total of 108 poultry processing plant samples were collected to test with conventional microbiology and qPCR. Sampling included cloacal swabs, swabs of transport crates (before and after the cleaning and disinfection process) and carcasses (after the chiller, cooled at 4°C and frozen at -12°C). Positivity in cloacal swabs indicated that poultry arrived contaminated at the slaughterhouse. Contamination in transport cages was substantially increased after the cleaning process, indicating that the process was ineffective. The detection of Campylobacter on carcasses was higher than that on cloacal swabs, which could indicate cross-contamination during the slaughtering process. Conventional microbiology and molecular methods revealed a prevalence of 69.4% and 43.5%, respectively. Lower detection by qPCR can be attributed to the high specificity of the kit and to biological components that could inhibit PCR reactions. Our results indicate that poultry arrive contaminated at the slaughterhouse and that contamination can increase during the slaughtering process due to cross-contamination. The isolation of Campylobacter in cooled and frozen carcasses corroborates the bacterial survival even at temperatures considered limiting to bacterial growth which are routinely used for food preservation.

Pathogens causing diarrhoea among Bangladeshi children with malignancy: Results from two pilot studies.

BACKGROUNDDiarrhoea is a frequent symptom in children with cancer, and occurs due to a composite effect of underlying disease and immunosuppression consequent to therapy, malnutrition, and non-infective aetiologies such as mucositis. In a large proportion of cases, the aetiology of diarrhoea remains unknown but is often attributed to multiple pathogens including parasites.AIMTo identify and describe the pathogens causing diarrhoea in Bangladeshi children with cancer.METHODSTwo cross-sectional pilot studies were conducted involving paediatric oncology patients with diarrhoea. Stool samples were collected from children who were hospitalised with or without being treated with chemotherapy during the study period, and had diarrhoea at any stage during their admission. In the first study, stool samples were tested by conventional microbiological methods and by polymerase chain reaction for parasites, and by immunoassays for Clostridium difficile. In the second study, conventional microbiology was conducted for bacteria and parasites including an enzyme-linked immunosorbent assay for Cryptosporidium antigen, and in a subset, immunoassays for Clostridium difficile.RESULTSIn the first study Giardia lamblia was detected in 68.5% of samples, Entamoeba histolytica in 13%, Cryptosporidium in 5.6%, non-toxigenic C. difficile in 22.4%, and other bacteria in 5.2%. In the second study, E. histolytica was detected in 10% of samples, Cryptosporidium in 4.3%, G. lamblia in 1.4%, C. difficile in 5.1%, and other bacteria in 5.7% of samples.CONCLUSIONThese pilot data suggest that parasites are important aetiologies of diarrhoea in Bangladeshi children with malignancy. While molecular diagnostic tools detect an array of stool pathogens with greater sensitivity, conventional diagnostic methods are also useful.

Bioprospección de bacterias ácido lácticas productoras de biofloculante aisladas de jugo de caña residual

The objective was to isolate bioflocculant-producing lactic acid bacteria (LAB) from residual sugarcane juice. For this reason, sugarcane juice samples were obtained from 15 randomly sampled residual sugarcane stalk samples. The isolation of LAB was then carried out by means of conventional microbiology techniques, using Agar Mayeux, Sandine and Elliker (MSE) culture medium at pH 7.2 and an incubation time of 30°C for 48 hours. Subsequently, pure cultures were made from the characteristic colonies of Leuconostoc mesenteroides (rubbery, viscous, translucent and creamy colonies) for their biochemical identification according to Bergey’s Manual of Bacteriological Determination. The identification and selection of L. mesenteroides subsp. mesenteroides was carried out according to the Kappa coefficient statistical method, with the purpose of using it in the production of dextran (bioflocculant) in an aerated-agitated bioreactor (Aplikon brand). The purity of the dextran was determined using the FT-IR technique, which was compared with the spectrum of pure dextran produced by the strain NRRL P-640. Four strains of Leuconostoc mesenteroides, LM (01-04) were isolated, of which the strain LM03 was identified as L. meenteroides subsp. mesenteroides. The dextran values produced by LM03 were 26.87 g/L at 80 hours (maximum concentration) and 2.61 g/L at 4 hours (minimum concentration). The dextran produced by LM03 is pure according to FT-IR analysis. In conclusion, it was possible to isolate BAL L. mesenteroides subsp. mesenteroides (strain LM03), which had the capacity to produce dextran, which can be used as a bioflocculant with different biotechnological and industrial uses.

One and Half Million Hematopoietic Stem Cell Transplants (HSCT). Dissemination, Trends and Potential to Improve Activity By Telemedicine from the Worldwide Network for Blood and Marrow Transplantation (WBMT)

HSCT is the only curative option for many malignant and non-malignant diseases. The WBMT was founded as an umbrella organization of societies involved in cellular therapies with the mission of promoting excellence in HSCT. As a non-governmental organization in official relation with the World Health Organization (WHO), the WBMT assisted in the founding of regional societies (Latin America Blood and Marrow Transplantation Group and African Blood and Marrow Transplantation Group), performs global activity surveys, conducts workshops and provides expert support for programs in evolving countries. In this retrospective evaluation we analyzed worldwide activity trends in HSCT up to the year 2016 and evaluated possibilities of improving availability of HSCT by the use of telemedicine. Methods: HSCT activity was collected annually from member societies, national registries and individual centers including donor type (allogeneic/autologous), stem cell source (bone marrow/peripheral blood stem cells/cord blood) and indications for transplant. Transplant rates (TR) were calculated as HSCT/10 million inhabitants without adjustment for patients transplanted in a country other than that of primary residence. Country team density (TD) was defined as teams/10 million inhabitants. Workshops were organized in a number of locations where there was little or no HSCT activity or where improvement in one or more aspect of local or regional HSCT activity was requested, including Vietnam, Brazil, China, South Africa and Morocco. Other countries were paired with established centers using WBMT affiliated partners. In two countries, a pilot program was established involving a 6 month physician training in a JACIE/FACT accredited center followed by daily telemedicine-guided supervision of clinical activities. Results: From 1957-2016 a total of 1,298,897 HSCT (57.1% autologous) procedures were collected. By the end of 2016, HSCT activity was reported from 87 of the 195 WHO member states. A total of 89,070 HSCT from 1662 centers was reported in 2016. Assuming a frequency of 84,000/year, 1.5 million HSCT will be reached in 2019, only 7 years after the 1 million report in 2012 (Figure 1). The global activity/year increased continuously from 10,000/year in 1991 to 82,718 first HSCT/year in 2016 with a global increase of >7% (7.0% in autologous and 7.8% in allogeneic HSCT). As in previous years, slightly more autologous (53.5%) than allogeneic and more related (53,6%) than unrelated HSCT were reported. The further increase in related HSCT was caused mainly by an increase of non-identical family donors (39.5% of related HSCT). Increase in activities according to regions is given in Figure 2. TR and TD varied according to region and are highest in Nord America with 511.2 TR, followed by Europe with 390.9 TR, Latin America with 63.9 TR, APBMT with 46.2 TR and Africa/EMRO with 32.8 TR. In contrast, TD was highest in Europe (7.5 TD) followed by Nord America (6.0 TD), APBMT (1.9 TD), Latin America (1.9 TD) and Africa/EMRO (0.4 TD). Commonest indications were lymphoproliferative diseases for autologous and leukemia for allogeneic HSCT and continue to rise (Figure 3 autologous and Figure 4 allogeneic HSCT). Graft source were predominantly peripheral blood in autologous (99.7%) and 65% in unrelated HSCT, while umbilical cord blood as a stem cell source (13.8% of all unrelated) declined. More than 150 HSCT were performed in one country and one center without activities using daily telemedicine-guided supervision. In conclusion, the global distribution and activities are increasing continuously by more than 7,0% per year with numbers currently running at app. 90,000/year. Of note is the increase of haploidentical HSCT activity, while the use of umbilical cord blood HSCT continues to decrease. TR data show significant gaps between regions. Supervisory telemedicine is a powerful tool to overcome lack of experience and establish JACIE/FACT compatible new programs with collateral benefits for conventional hematology, blood banking, microbiology and virology. Abbreviations: EUR, Europe; AMR/PHA, America; SEAR/WPR, South-East Asia/Western Pacific; AFR/EMRO, African/Eastern Mediterranean. Disclosures Niederwieser: Daichii: Speakers Bureau; Cellectis: Consultancy. Atsuta:Kyowa Kirin Co., Ltd: Honoraria; Chugai Pharmaceutical Co., Ltd.: Honoraria; Mochida Pharmaceutical Co. Ltd: Honoraria; Janssen Paharmaceutical K.K.: Honoraria. Worel:Sanofi Genzyme, Malinckrodt Therakos: Research Funding; Jazz, Sanofi, Celgene, Novartis, Malinckrodt Therakos: Honoraria; Sanofi Genzyme, Malinckrodt Therakos: Speakers Bureau. Galeano:Szabo SA: Other: (Equity interest). Novitzky:Astellas, Roche: Consultancy. Szer:Prevail Therapeutics: Honoraria, Other: Travel, Research Funding; Novartis: Honoraria, Other: Travel, Research Funding; MSD: Honoraria, Other: Travel, Research Funding; Celgene: Honoraria, Other: Travel, Research Funding; Amgen: Honoraria, Other: Travel, Research Funding; Alexion: Honoraria, Other: Travel, Research Funding; Sanofi: Honoraria, Other: Travel, Research Funding; Takeda: Honoraria, Other: Travel, Research Funding; Pfizer: Honoraria, Other: Travel, Research Funding. Kröger:Celgene: Honoraria, Research Funding; DKMS: Research Funding; JAZZ: Honoraria; Medac: Honoraria; Neovii: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Riemser: Research Funding; Sanofi-Aventis: Research Funding. Weisdorf:Incyte: Research Funding; Pharmacyclics: Consultancy; Fate Therapeutics: Consultancy. Pasquini:Amgen: Consultancy; BMS: Research Funding; Medigene: Consultancy; Pfizer: Other: Advisory Board; Novartis: Research Funding; Kit Pharma: Research Funding.

A comparative assessment of conventional and molecular methods, including MinION nanopore sequencing, for surveying water quality

Nucleic acid based techniques, such as quantitative PCR (qPCR) and next generation sequencing (NGS), provide new insights into microbial water quality, but considerable uncertainty remains around their correct interpretation. We demonstrate, for different water sources in informal settlements in the Kathmandu Valley, Nepal, significant Spearman rank correlations between conventional and molecular microbiology methods that indicate faecal contamination. At family and genera level, 16S rRNA amplicon sequencing results obtained with the low-cost, portable next generation sequencer MinION from Oxford Nanopore Technologies had significant Spearman rank correlations with Illumina MiSeq sequencing results. However, method validation by amplicon sequencing of a MOCK microbial community revealed the need to ascertain MinION sequencing results for putative pathogens at species level with complementary qPCR assays. Vibrio cholerae hazards were poorly associated with plate count faecal coliforms, but flagged up by the MinION screening method, and confirmed by a qPCR assay. Plate counting methods remain important to assess viability of faecal coliforms in disinfected water sources. We outline a systematic approach for data collection and interpretation of such complementary results. In the Kathmandu Valley, there is high variability of water quality from different sources, including for treated water samples, illustrating the importance of disinfection at the point of use.