Presence Of Multiple Pathogens Research Articles

Enteric Pathogens Detected in Children under Five Years Old Admitted with Diarrhea in Moshi, Kilimanjaro, Tanzania.

Despite the availability and wide coverage of rotavirus vaccinations in Tanzania, there is still a significant number of diarrhea cases being reported, with some patients requiring hospital admission. We investigated diarrhea-causing pathogens and determined the effect of co-infection on clinical symptoms. Total nucleic acid was extracted from archived stool samples (N = 146) collected from children (0-59 months) admitted with diarrhea in health facilities in Moshi, Kilimanjaro. Pathogen detection was performed using the quantitative polymerase chain reaction with custom TaqMan Array cards. The Poisson model was used to determine the effect of co-infection on clinical presentation during admission. Of all the participants, 56.85% were from rural Moshi with a median age of 11.74 months (IQR: 7.41-19.09). Vomiting (88.36%) and a fever (60.27%) were the most frequent clinical manifestations. At least one diarrhea-associated pathogen was detected in 80.14% (n = 117) of the study population. The most prevalent pathogens were rotavirus 38.36% (n = 56), adenovirus 40/41 19.86% (n = 29), Shigella/EIEC 12.33% (n = 18), norovirus GII 11.44% (n = 17) and Cryptosporidium 9.59% (n = 14). Co-infections were detected in 26.03% of the study population (n = 38). The presence of multiple pathogens in the stool samples of children with diarrhea indicates poor sanitation and may have significant implications for disease management and patient outcomes.

Probing Interkingdom Signaling Molecules via Liquid Extraction Surface Analysis-Mass Spectrometry.

Previously, metabolites diffused or secreted from microbial samples have been analyzed via liquid chromatography-mass spectrometry (LC-MS) approaches following lengthy extraction protocols. Here, we present a model system for growing biofilms on discs before utilizing rapid and direct surface sampling MS, namely, liquid extraction surface analysis, to study the microbial exometabolome. One of the benefits of this approach is its surface-specific nature, enabling mimicking biofilm formation in a way that the study of planktonic liquid cultures cannot imitate. Even though Pseudomonas aeruginosa (P. aeruginosa), Staphylococcus aureus (S. aureus), and Candida albicans (C. albicans) have been studied previously in isolation, very few studies consider the complexity of the interplay between these pathogens, which are commonly combined causative agents of infection. Our model system provides a route to investigate changes in the exometabolome, such as metabolites that become circulatory in the presence of multiple pathogens. Our results agree with previous reports showing that 2-alkyl-4(1H)-quinolone signal molecules produced by P. aeruginosa are important markers of infection and suggest that methods for monitoring levels of 2-heptyl-4-hydroxyquinoline and 2,4-dihydroxyquinoline, as well as pyocyanin, could be beneficial in the determination of causative agents in interkingdom infection including P. aeruginosa. Furthermore, studying changes in exometabolome metabolites between pqs quorum sensing antagonists in treated and nontreated samples suggests suppression of phenazine production by P. aeruginosa. Hence, our model provides a rapid analytical approach to gaining a mechanistic understanding of bacterial signaling.

Respiratory pathogens associated with intubated pediatric patients following hematopoietic cell transplant.

We describe organisms found in the respiratory tracts of a multicenter cohort of pediatric hematopoietic cell transplant (HCT) recipients with respiratory failure. Twelve centers contributed up to 25 pediatric allogeneic HCT recipients requiring mechanical ventilation for respiratory failure to a retrospective database. Positive respiratory pathogens and method of obtaining sample were recorded. Outcomes were assessed using Mann-Whitney U test or chi-squared analysis. Of the 222 patients in the database, ages 1month through 21years, 34.6% had a positive respiratory culture. 105 pathogens were identified in 77 patients; of those, 48.6% were viral, 34.3% bacterial, 16.2% fungal, and 1% parasitic. PICU mortality with a respiratory pathogen was 68.8% compared to 54.9% for those without a respiratory pathogen (P=.045). Those with a positive respiratory pathogen had longer PICU length of stay, 20days (IQR 14.0, 36.8) vs 15 (IQR 6.5, 32.0), P=.002, and a longer course of mechanical ventilation, 17days (IQR 10, 29.5) vs 8 (3, 17), P<.0001. Method of pathogen identification, type of pathogen, and the presence of multiple pathogens were not associated with changes in PICU outcomes. In this multicenter retrospective cohort of intubated pediatric post-HCT patients, there was high variability in the respiratory pathogens identified. Type of pathogen and method of detection did not affect PICU mortality. The presence of any organism leads to increased PICU mortality, longer PICU stay, and increased duration of mechanical ventilation suggesting that early detection and treatment of pathogens may be beneficial in this population.

2135. Streptococcus pneumoniae DNA (lytA) Detection in Clinical Samples Sent for a Respiratory Viral Polymerase Chain Reaction: Is There Bacterial-Viral Association?

BackgroundStreptococcus pneumoniae (SPNEU) is a major cause of community-acquired pneumonia and frequently complicates respiratory viral infections. Clinical differentiation of viral and bacterial respiratory tract infection can be difficult, as can predicting which patients with respiratory viral infection will develop bacterial infection. A SPNEU polymerase chain reaction (PCR) to specific DNA region lytA may be able to determine which patients with viral infections may develop bacterial infection.MethodsStored nucleic acid extracts from clinical samples collected and tested for respiratory viral PCR (RVP) in 2015 and 2016 were tested for SPNEU lytA DNA using standard laboratory procedure. Analyses of demographic data, RVP and SPNEU PCR result were performed to determine relevant associations.Results1581 stored clinical RVP samples were tested for SPNEU DNA with PCR to lytA, 1550 of these had complete RVP panel results available for analysis. RVP samples from patients 0–5 years old were more likely to have a viral or bacterial pathogen detected than > 5 years old (78% vs. 45%, P < 0.001). Of 1,550 samples analyzed with SPNEU PCR, 19% were positive for SPNEU, this was more likely in those 0–5 years old than > 5 years old (50% vs. 10%, P < 0.0001). In 0–5 years old, SPNEU was more frequently detected when multiple pathogens were detected on RVP vs. those with no pathogen (63% vs. 43%, P = 0.031). In > 5 years old, compared with no pathogen samples, the presence of multiple pathogens, any single pathogen, influenza, rhinovirus and Bordetella pertussis were significantly associated with higher SPNEU positivity rates. Median SPNEU PCR DNA load was higher in multiple pathogen and single pathogen samples than in no pathogen RVP samples.ConclusionWe have demonstrated an association between common respiratory pathogens and detection of SPNEU DNA via PCR. This association is strongest in samples with multiple RVP pathogens, suggesting additional nasopharyngeal inflammation may contribute to SPNEU presence in the nasopharynx. Previous data have focused on those 0–5 years old, we have demonstrated an SPNEU-viral association in those > 5 years old. This tool may be clinically useful to determine which individuals with viral respiratory tract infection will progress to bacterial pneumonia and warrants further investigation.Disclosures All authors: No reported disclosures.

Investigation of suspected Newcastle disease (ND) outbreaks in Egypt uncovers a high virus velogenic ND virus burden in small-scale holdings and the presence of multiple pathogens

ABSTRACTHighly contagious Newcastle disease (ND) is associated with devastating outbreaks with highly variable clinical signs among gallinaceous birds. In this study we aimed to verify clinical ND suspicions in poultry holdings in Egypt suffering from respiratory distress and elevated mortality, comparing two groups of ND-vaccinated poultry holdings in three governorates. Besides testing for Newcastle disease virus (NDV), samples were screened for infectious bronchitis virus (IBV) and avian influenza virus (AIV) by RT-qPCR as well as by non-directed cell-culture approach on LMH-cells. Virulent NDV was confirmed only in group A (n = 16) comprising small-scale holdings. Phylogenetic analysis of the fusion protein gene of 11 NDV-positive samples obtained from this group assigned all viruses to genotype 2.VIIb and point to four different virus populations that were circulating at the same time in one governorate, indicating independent epidemiological events. In group B, comprising large commercial broiler farms (n = 10), virulent NDV was not present, although in six farms NDV vaccine-type virus (genotype 2.II) was detected. Besides, in both groups, co-infections by IBV (n = 10), AIV H9 (n = 3) and/or avian reovirus (ARV) (n = 5) and avian astrovirus (AastVs) (n = 1) could be identified. Taken together, the study confirmed clinical ND suspicion in small scale holdings, pointing to inefficient vaccination practices in this group A. However, it also highlighted that, even in an endemic situation like ND in Egypt, in cases of suspected ND vaccine failure, clinical ND suspicion has to be verified by pathotype-specific diagnostic tests.RESEARCH HIGHLIGHTSVelogenic NDV circulates in small-scale poultry holdings in Egypt.Viral transmission occurred among neighbouring farms and over long distances.Co-infections with multiple pathogens were identified.Pathotype specific diagnostic tests are essential to verify ND suspicions.

Pathogen Clearance and New Respiratory Tract Infections Among Febrile Children in Zanzibar Investigated With Multitargeting Real-Time Polymerase Chain Reaction on Paired Nasopharyngeal Swab Samples.

New molecular methods have revealed frequent and often polymicrobial respiratory infections in children in low-income settings. It is not known whether presence of multiple pathogens is due to prolonged infections or to frequent exposure. The aim of this study was to analyze short-term pathogen clearance from nasopharynx and the rate of new respiratory tract infections in febrile preschool children. Children (n = 207) with uncomplicated acute febrile illness 2-59 months of age presenting to a health center in Zanzibar, Tanzania, April-July 2011, were included. Paired nasopharyngeal swab samples, collected at enrolment and after 14 days, were analyzed by multiple real-time polymerase chain reaction for Adenovirus, bocavirus, Bordetella pertussis, Chlamydophila pneumoniae, Coronaviruses, Enterovirus, influenza A and B virus, metapneumovirus, measles virus, Mycoplasma pneumoniae, parainfluenza virus, Parechovirus, respiratory syncytial virus and Rhinovirus. An age-matched and geographically matched healthy control group (n = 166) underwent nasopharyngeal sampling on 1 occasion. At baseline, 157/207 (76%) patients had at least 1 pathogen detected, in total 199 infections. At follow-up (day 14), 162/199 (81%) of these infections were not detected, including >95% of the previously detected infections with Enterovirus, influenza A virus, influenza B virus, metapneumovirus or parainfluenza virus. Still 115 (56%) children were positive for at least 1 pathogen at follow-up, of which 95/115 (83%) were not found at baseline. Detection of influenza B on day 14 was significantly associated with fever during follow-up. The results suggest that children with acute febrile illness in Zanzibar rapidly clear respiratory tract infections but frequently acquire new infections within 14 days.

Rapid identification of fungi in culture-negative clinical blood and respiratory samples by DNA sequence analyses

BackgroundClinical diagnoses of fungal infections often rely upon culture techniques followed by microscopic examination of positive cultures and histopathological specimens. Culturing of microorganisms is prone to false negatives, while microscopy methods can be complicated by atypical phenotypes and organisms that are morphologically indistinguishable in tissues. Delays in diagnoses (or the lack thereof) and inaccurate identification of infectious organisms contribute to increased morbidity and mortality in patients.MethodsTwo-hundred randomized, heterogeneous patient blood and respiratory samples that were culture-negative were tested using polymerase chain reaction (PCR) amplification of internal transcribed spacer regions of ribosomal RNA genes utilizing panfungal primers. Amplicons were sequenced, subjected to sequence similarity searches, and compared using phylogenetic analyses.ResultsThirteen fungal sequences were detected in three whole-blood samples and nine respiratory samples. Bioinformatic analyses were performed which indicated the presence of multiple pathogens and potential pathogens.ConclusionsThe results from this pilot study demonstrate the utility of PCR assays and sequence analyses in clinical tests for fungi to facilitate rapid diagnosis and appropriate treatments to deal with the false negatives from culture results.

Etiology of community acquired pneumonia among children in India: prospective, cohort study.

BackgroundChildhood community acquired pneumonia (CAP) is a significant problem in developing countries, and confirmation of microbial etiology is important for individual, as well as public health. However, there is paucity of data from a large cohort, examining multiple biological specimens for diverse pathogens (bacteria and viruses). The Community Acquired Pneumonia Etiology Study (CAPES) was designed to address this knowledge gap.MethodsWe enrolled children with CAP (based on WHO IMCI criteria of tachypnea with cough or breathing difficulty) over 24 consecutive months, and recorded presenting symptoms, risk factors, clinical signs, and chest radiography. We performed blood and nasopharyngeal aspirate (NPA) bacterial cultures, and serology (Mycoplasma pneumoniae, Chlamydophila pneumoniae). We also performed multiplex PCR for 25 bacterial/viral species in a subgroup representing 20% of the cohort. Children requiring endotracheal intubation underwent culture and PCR of bronchoalveolar lavage (BAL) specimens.FindingsWe enrolled 2345 children. NPA and blood cultures yielded bacteria in only 322 (13.7%) and 49 (2.1%) children respectively. In NPA, Streptococcus pneumoniae (79.1%) predominated, followed by Haemophilus influenzae (9.6%) and Staphylococcus aureus (6.8%). In blood, S. aureus (30.6%) dominated, followed by S. pneumoniae (20.4%) and Klebsiella pneumoniae (12.2%). M. pneumoniae and C. pneumoniae serology were positive in 4.3% and 1.1% respectively. Multiplex PCR in 428 NPA specimens identified organisms in 422 (98.6%); of these 352 (82.2%) had multiple organisms and only 70 (16.4%) had a single organism viz. S. pneumoniae: 35 (50%), Cytomegalovirus (CMV): 13 (18.6%), Respiratory Syncytial Virus (RSV): 9 (12.9%), other viruses: 6 (8.7%), S. aureus: 5 (7.1%), and H. influenzae: 2 (2.9%). BAL PCR (n = 30) identified single pathogens in 10 (S. pneumoniae–3, CMV–3, S. aureus–2, H. influenzae–2) and multiple pathogens in 18 children. There were 108 (4.6%) deaths. The pattern of pathogens identified did not correlate with pneumonia severity or mortality.ConclusionsThe majority of children with CAP have multiple pathogens (bacteria and viruses). S. pneumoniae and S. aureus predominate in NPA and blood respectively. CMV and RSV were the dominant respiratory viruses in NPA and BAL. The presence of multiple pathogens, especially organisms associated with nasopharyngeal carriage, precludes confirmation of a causal relationship in most cases.

Eukaryotic pathogens (Chytridiomycota and Oomycota) infecting marine microphytobenthic diatoms - a methodological comparison.

Using sediment samples from the Solthörn tidal flat (southern North Sea, Germany), collected in bi-weekly intervals from June to July 2012, a range of qualitative and quantitative screening methods for oomycete and chytrid pathogens infecting benthic diatoms were evaluated. Pre-treatment of sediment samples using short ultrasound pulses and gradient centrifugation, in combination with CalcoFluor White, showed the best results in the visualization of both pathogen groups. The highest number of infected benthic diatoms was observed in mid July (5.8% of the total benthic diatom community). Most infections were caused by chytrids and, in a few cases, oomycetes (Lagenisma Drebes (host: Coscinodiscus radiatus Ehrenberg) and Ectrogella Zopf (hosts: Dimeregramma minor in Pritchard and Gyrosigma peisonis). Among the chytrids, sporangium morphology indicated the presence of five different morphotypes, infecting mainly epipelic taxa of the orders Naviculales (e.g., Navicula digitoradiata) and Achnanthales (e.g., Achnanthes brevipes Agardh). The presence of multiple pathogens in several epipelic diatom taxa suggests a significant role for fungal parasitism in affecting microphytobenthic diatom succession.

Respiratory syncytial and parainfluenza type 3 viruses coexisting with Pasteurella multocida and Mannheimia hemolytica in acute pneumonias of neonatal alpacas

The etiopathogenesis of acute pneumonia, the second most important cause of mortality among neonatal alpacas in Peru, is still poorly understood. The objective of this study was to characterize gross and histopathology lesions, as well as to identify viruses [parainfluenza type 3 (PI-3) and/or bovine respiratory syncytial (BRS)] by direct inmunofluorescence test, and isolate bacteria [ Pasteurella multocida (Pm) and/or Mannheimia haemolytica (Mh)] from 24 fatal acute pneumonia cases of alpaca neonates 7–39 days old. At necropsy the gross lesions corresponded to moderate purulent focal bronchopneumonia or severe necrotic purulent fibrinous ( n = 13), and moderate to severe pulmonary congestion and/or pulmonary edema ( n = 11). Histopathological analysis revealed: acute severe and diffuse necrotizing, fibrinous, suppurative bronchopneumonia ( n = 3), acute mild to moderate and focally diffuse suppurative bronchopneumonia ( n = 10) and acute moderate to severe diffuse congestion and pulmonary edema ( n = 11). Among these 24 fatal cases, 22 yielded virus identification and/or bacterial isolation. Eight of these 22 cases were positive for only one pathogen (5 for viruses and 3 for bacteria), 10/22 were positive for 2 pathogens RSV plus bacteria ( n = 7), PI-3 plus bacteria ( n = 2) and 1 for both bacteria and 4/22 were positive for 3 pathogens [RSV, PI-3 plus bacteria ( n = 3) and PI-3 plus both bacteria ( n = 1)]. Among the affected lung tissues, virus was identified 19 times (13 positive for RSV, 9 for PI-3, and 3/19 for both viruses) whereas bacteria were isolated 14 times [ P. multocida ( n = 8), M. haemolytica ( n = 6), and both bacteria ( n = 2)]. The presence of multiple pathogens was observed in 14/22 positive cases with an observation of virus–bacteria association in 13/14 of the cases. The coexistence of RSV-Pm was the most frequently observed (6/13) followed by the simultaneous presence of RSV-Mh (4/13) and PI-3 Pm or Mh (4/13). These results appear to indicate that acute pneumonia in alpaca neonates may well result from virus and bacterial interactions in a similar way to pneumonic lesions of other ruminants.

Sudden deaths and colony population decline in Greek honey bee colonies

During June and July of 2009, sudden deaths, tremulous movements and population declines of adult honey bees were reported by the beekeepers in the region of Peloponnesus (Mt. Mainalo), Greece. A preliminary study was carried out to investigate these unexplained phenomena in this region. In total, 37 bee samples, two brood frames containing honey bee brood of various ages, eight sugar samples and four sugar patties were collected from the affected colonies. The samples were tested for a range of pests, pathogens and pesticides. Symptomatic adult honey bees tested positive for Varroa destructor, Nosema ceranae, Chronic bee paralysis virus (CBPV), Acute paralysis virus (ABPV), Deformed wing virus (DWV), Sacbrood virus (SBV) and Black queen cell virus (BQCV), but negative for Acarapis woodi. American Foulbrood was absent from the brood samples. Chemical analysis revealed that amitraz, thiametoxan, clothianidin and acetamiprid were all absent from symptomatic adult bees, sugar and sugar patty samples. However, some bee samples, were contaminated with imidacloprid in concentrations between 14 ng/g and 39 ng/g tissue. We present: the infection of Greek honey bees by multiple viruses; the presence of N. ceranae in Greek honey bees and the first record of imidacloprid (neonicotonoid) residues in Greek honey bee tissues. The presence of multiple pathogens and pesticides made it difficult to associate a single specific cause to the depopulation phenomena observed in Greece, although we believe that viruses and N. ceranae synergistically played the most important role. A follow up in-depth survey across all Greek regions is required to provide context to these preliminary findings.

Salivary interleukin‐1β concentration and the presence of multiple pathogens in periodontitis

This study aimed to find salivary enzymes and/or cytokines that would reflect periodontitis, alone or in combination with salivary microbial markers. The salivary concentrations of elastase, lactate dehydrogenase, interleukin-1beta (IL-1beta), interleukin-6, and tumour necrosis factor-alpha, and the presence of five periodontal pathogens, Aggregatibacter actinomycetemcomitans, Porphyromonas gingivalis, Prevotella intermedia, Tannerella forsythia, and Treponema denticola, were analysed from salivary specimens of 165 subjects, a subpopulation of Health 2000 Health Examination Survey in Finland; 84 of the subjects had probing pocket depth (PPD) of > or =4 mm at 14 or more teeth (the advanced periodontitis group), while 81 subjects had no teeth with PPD of > or =4 mm (the control group). All subjects had at least 20 teeth and no systemic diseases. Among the salivary cytokines and enzymes tested, IL-1beta was the only biomarker associated with periodontitis. An association was also found with the presence of multiple periodontal pathogens. Salivary IL-1beta and the presence of multiple periodontal pathogens were associated with periodontitis at the same magnitude, when they were in the logistic regression model individually or together. We suggest that salivary IL-1beta and the presence of multiple periodontal pathogens in saliva should be studied more thoroughly as markers of periodontitis.