Aquatic and terrestrial cyanobacteria produce methane.

Evidence is accumulating to challenge the paradigm that biogenic methanogenesis, considered a strictly anaerobic process, is exclusive to archaea. We demonstrate that cyanobacteria living in marine, freshwater, and terrestrial environments produce methane at substantial rates under light, dark, oxic, and anoxic conditions, linking methane production with light-driven primary productivity in a globally relevant and ancient group of photoautotrophs. Methane production, attributed to cyanobacteria using stable isotope labeling techniques, was enhanced during oxygenic photosynthesis. We suggest that the formation of methane by cyanobacteria contributes to methane accumulation in oxygen-saturated marine and limnic surface waters. In these environments, frequent cyanobacterial blooms are predicted to further increase because of global warming potentially having a direct positive feedback on climate change. We conclude that this newly identified source contributes to the current natural methane budget and most likely has been producing methane since cyanobacteria first evolved on Earth.

Introducing rotavirus vaccine to the Palestinian territories: the role of public-private partnerships.

BACKGROUND: Introducing childhood immunization poses challenges in environments of societal fragility. The Palestinian territories (Pt) are considered 'fragile' because of their lack of political, economic and territorial sovereignty. Poverty is rife, infant mortality high, and diseases associated with overcrowding widespread. Under these circumstances the Rostropovich Vishneskaya Foundation (RVF) has assembled a network of public and private stakeholders to introduce a country-wide rotavirus immunization program. METHODS: The incidence of diarrhea was determined for 18 months before and 18 months after the introduction of rotavirus vaccine among all children younger than 5 years presenting to outpatient clinics in Gaza with three or more loose stools per day. Simultaneously the prevalence of rotavirus was established by rotavirus antigen detection in stool samples collected from children younger than 3 years at Caritas Baby Hospital in Bethlehem during the corresponding time periods. RESULTS: Within 12 months 97.4% immunization coverage was achieved. The incidence of diarrhea dropped by 32.2%, while the prevalence of rotavirus in stool samples decreased by 64.6% throughout the following year. CONCLUSION: In environments of economic or political instability private-public partnerships for the introduction of comprehensive vaccination programs can work based on close collaboration, shared vision, flexibility and inter-organizational trust.

The immunomodulator, ammonium trichloro[1,2-ethanediolato-O,O']-tellurate, suppresses the propagation of herpes simplex virus 2 by reducing the infectivity of the virus progeny.

Persistent investigations for the identification of novel anti-herpetic drugs are being conducted worldwide, as current treatment options are sometimes insufficient. The immunomodulator, ammonium trichloro[1,2­ethanediolato­O,O']­tellurate (AS101), a non­toxic tellurium (Ⅳ) compound, has been shown to exhibit anti­viral activity against a variety of viruses in cell cultures and in animal models. In the present study, the anti­viral activity of AS101 against herpes simplex virus (HSV)­1 and 2 was investigated in vitro. The results demonstrated that AS101 significantly restricted HSV­2-induced plaque formation and reduced the infectivity of the HSV­2 yield, while HSV­1 was affected to a lesser extent. The incubation of mature HSV­1 and HSV­2 viruses with AS101 had no effect on viral infectivity, indicating that the compound interrupts de novo viral synthesis. The addition of AS101 at up to 9 h post­infection had almost the same effect as did the addition of the drug together with the virus (it maintained 80% of its total anti­viral capacity). Quantitative PCR and immunofluoresence staining of viral structural proteins revealed that the viral DNA and protein synthesis stages were not interrupted by the administration of AS101. By contrast, in the presence of the compound, significantly fewer viable viruses (≥2 log reduction) were recovered from the AS10­treated cell cultures. Of note, when we determined the viability of the intracellular virus, formed in the presence of the compound, a less severe (≤1 log) effect was observed. Taken together, these data strongly suggest that AS101 primarily interferes with late stages of viral replication, such as viral particle envelopment or egress, leading to the production of a defective virus progeny.

Development and validation of a real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay for investigation of wild poliovirus type 1-South Asian (SOAS) strain reintroduced into Israel, 2013 to 2014.

In February 2013, wild poliovirus type 1 (WPV1) was reintroduced into southern Israel and resulted in continuous silent circulation in the highly immune population. As a part of the public health emergency response, a novel real time quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assay was developed, to allow for the sensitive and specific detection of the circulatingWPV1-South Asian (SOAS) strain. Specific primers and probes derived from the VP-1 region were designed, based on sequenced sewage isolates, and used to simultaneously amplify this WPV1-SOAS sequence together with bacteriophage MS-2 as internal control. High titre WPV1-SOAS stock virus was used for assay optimisation and 50 processed sewage samples collected from southern Israel and tested by reference culture based methods were used for analytical validation of the assay's performance. The limit of detection of the multiplex qRT-PCR (SOAS/MS-2) assay was 0.1 plaque-forming unit (pfu)/reaction (20 pfu/mL) for WPV1-SOAS RNA with 100% sensitivity, specificity, positive and negative predictive values when compared to the culture based method. The turnaround time was rapid, providing results for environmental samples within 24 to 48 hours from completion of sewage processing, instead of five to seven days by culture-based analysis. Direct sewage testing by qRT-PCR assay proved to be a useful tool for rapid detection and environmental surveillance of WPV1-SOAS circulating strain during emergency response. Application of the approach for detection of WPV1-SOAS in stool samples obtained during acute flaccid paralysis (AFP) surveillance or field surveys should be further evaluated.

Molecular epidemiology of silent introduction and sustained transmission of wild poliovirus type 1, Israel, 2013.

Poliovirus vaccine coverage in Israel is over 90%. The last nine birth cohorts have been vaccinated exclusively with inactivated polio vaccine (IPV). However, between February and July 2013 type 1 wild poliovirus (WPV1) was detected persistently in 10 and intermittently in 8 of 47 environmental surveillance sites in southern and central Israel and in 30 stool samples collected during July from healthy individuals in southern Israel. We report results of sequence and phylogenetic analyses of genes encoding capsid proteins to determine the source and transmission mode of the virus. WPV1 capsid protein 1 nucleotide sequences were most closely related to South Asia (SOAS) cluster R3A polioviruses circulating in Pakistan in 2012 and isolated from Egyptian sewage in December 2012. There was no noticeable geographical clustering within WPV1-positive sites. Uniform codon usage among isolates from Pakistan, Egypt and Israel showed no signs of optimisation or deoptimisation. Bayesian phylogenetic time clock analysis of the entire capsid coding region (2,643 nt) with a 1.1% evolutionary rate indicated that Israeli and Egyptian WPV1-SOAS lineages diverged in September 2012, while Israeli isolates split into two sub-branches after January 2013. This suggests one or more introduction events into Israel with subsequent silent circulation despite high population immunity.

Intensified environmental surveillance supporting the response to wild poliovirus type 1 silent circulation in Israel, 2013.

An emergency response was triggered by recovery of wild poliovirus type 1 (WPV1) of the South Asia (SOAS) lineage from sewage in southern Israel in April 2013 during routine environmental surveillance. Public health risk assessment necessitated intensification of environmental surveillance in order to facilitate countrywide monitoring of WPV1-SOAS circulation. This involved increasing sampling frequency and broadening the geographical area, for better coverage of the population at risk, as well as modifying sewage testing algorithms to accommodate a newly developed WPV1-SOAS-specific quantitative real-time RT-PCR assay for screening of RNA extracted directly from sewage concentrates, in addition to standard virus isolation. Intensified surveillance in 74 sites across Israel between 1 February and 31 August 2013 documented a sustained high viral load of WPV1-SOAS in sewage samples from six Bedouin settlements and two cities with Jewish and Arab populations in the South district. Lower viral loads and intermittent detection were documented in sampling sites representing 14 mixed communities in three of the five health districts in central and northern Israel. Environmental surveillance plays a fundamental role in routine monitoring of WPV circulation in polio-free countries. The rapid assay specific for the circulating strain facilitated implementation of intensified surveillance and informed the public health response and decision-making.

Legionella pneumophila contamination of a dental unit water line system in a dental teaching centre.

OBJECTIVE: This study aimed to evaluate the extent of Legionella pneumophila contamination in a dental unit water line (DUWL) at a Dental Teaching Centre in Jordan. METHODS: Ten dental units were sampled from each teaching clinic, namely conservative dentistry, periodontology and prosthodontics. Samples were collected from the air/water syringe, high-speed hand piece and water cup filler. Sampling time was at the beginning of the working day (before the dental unit was used), after 2 min of flushing, and at midday. RESULTS: Legionella pneumophila counts ranged between 0 and 8.35 x 10(3) (CFU ml(-1)). Legionella pneumophila was detected in 86.7% of the dental units at the beginning of the working day, 40% after 2 min flushing and 53.3% at midday. The highest L. pneumophila counts were found at the beginning of the working day which were reduced by flushing the waterlines. The conservative dentistry clinic had the highest contamination level followed by the periodontology and prosthodontics clinics (P < 0.05). The rate of contamination can be ascribed to the dental procedures performed in the clinics, the degree of using the hand pieces, and water softening and heating. CONCLUSIONS: The difficulty of completely eliminating micro-organism contaminating water used for dental treatment and the resulting biofilm suggest that flushing of DUWL can be a first solution in reducing L. pneumophila counts, while the incorporation of a disinfection method is highly recommended. Water heating and softening should be considered in practicing dentistry as factors that may aid in L. pneumophila proliferation inside the DUWL.

The presence of Pseudomonas aeruginosa in the dental unit waterline systems of teaching clinics.

OBJECTIVE: The objective of this study was to evaluate the extent of Pseudomonas aeruginosa contamination of Dental Unit Water (DUW) at a Dental Teaching Center in Jordan. METHODS: Water samples were collected from 30 dental units, 10 from each of three teaching clinics, namely conservative dentistry, periodontology, and prosthodontics. Samples were collected from the outlet of the air/water syringe, high-speed handpiece and water cup filler, at the beginning of the working day (before use), after 2 min flushing, and at midday. RESULTS: P. aeruginosa was detected in 86.7% (26/30) of the dental units at the beginning of the working day, and in 73.3% (22/30) after 2 min of flushing and at midday. Conservative dentistry units had the highest counts, followed by periodontology and prosthodontics (P<0.05). Overall, the highest counts (log10 count CFU ml-1) were at the beginning of the working day (1.38+/-1.05), and the lowest counts after flushing for 2 min (1.10+/-1.03), and higher numbers were seen again at midday (1.15+/-1.04) (P<0.05). CONCLUSIONS: 86.7% of the dental units were contaminated with P. aeruginosa, the conservative dentistry units had the highest amount of contamination. Flushing the DUW for 2 min significantly reduced the counts of P. aeruginosa.

Isolation and characterization of West Nile virus from the blood of viremic patients during the 2000 outbreak in Israel.

We report the isolation of West Nile (WN) virus from four patient serum samples submitted for diagnosis during an outbreak of WN fever in Israel in 2000. Sequencing and phylogenetic analysis revealed two lineages, one closely related to a 1999 New York isolate and the other to a 1999 Russian isolate.

Evaluation of the Prodesse Hexaplex multiplex PCR assay for direct detection of seven respiratory viruses in clinical specimens.

We evaluated the Hexaplex assay (Prodesse, Waukesha, WI) for the detection of 7 respiratory viruses (influenza A and B, parainfluenza 1-3, and respiratory syncytial virus [RSV] A and B). The Hexaplex assay was performed on 300 respiratory samples during the 1999-2000 respiratory virus season. Results of this assay were compared with shell vial cell culture and/or direct fluorescent antibody stain. The overall sensitivity and specificity of the assay were 96.6% and 94.1%, respectively. The respective sensitivity and specificity of the Hexaplex assay for detection of specific virus groups were as follows: influenza A, 98.6% and 97.8%; influenza B, 100% and 100%; and for parainfluenza viruses (1-3), 100% and 99.1%. The assay did not perform as well with patients infected with RSV: sensitivity and specificity were 91.0% and 98.6%, respectively. There are 2 major drawbacks to this assay: it is technically demanding (3-4 hours hands-on time), and it is expensive ($80-$90 direct cost). Nevertheless, because of the excellent sensitivity and specificity, the Hexaplex assay may be valuable in the diagnosis of respiratory viral infections in immunocompromised patients.

Invasive serotype a Haemophilus influenzae infections with a virulence genotype resembling Haemophilus influenzae type b: emerging pathogen in the vaccine era?

OBJECTIVE: Haemophilus influenzae type b causes severe disease in nonimmune infants and young children; other serotypes are uncommon pathogens and thought to have low virulence. Some have hypothesized that with the virtual elimination of H influenzae type b, other serotypes might acquire virulence traits and emerge as important pathogens of children. We describe the clinical, epidemiologic, and molecular biologic features of 5 cases of severe disease attributable to Haemophilus influenzae type a. METHODS: After observing 4 cases of invasive disease caused by H influenzae type a, we reviewed microbiology records at 3 reference laboratories that perform all serotyping in Utah and surveillance databases. Strains of H influenzae type a and control strains were examined by Southern blotting with the use of the cap probe pUO38 and by pulsed-field gel electrophoresis. The putative virulence mutation, the IS1016-bexA deletion, was detected by polymerase chain reaction amplification and sequencing. RESULTS: During a 10-month period, we observed 5 children with severe invasive disease caused by H influenzae type a. No isolates of H influenzae type a had been submitted to the reference laboratories between 1992 and 1998. The median age of patients was 12 months (range: 6-48 months). Four of 5 had meningitis and bacteremia; 1 had purpura fulminans. Three isolates, representing 1 of 2 pulsed-field gel electrophoresis patterns, contained the IS1016-bexA deletion and were associated with particularly severe disease. CONCLUSIONS: We describe an unusual cluster of severe disease caused by H influenzae type a that resembles the clinical and epidemiologic features of H influenzae type b disease. Our data support the hypothesis that the IS1016-bexA deletion may identify more virulent strains of H influenzae. Haemophilus influenzae, epidemiology, virulence, serotyping, pathogenicity.

Comparison of three transport systems (Starplex StarSwab II, the new Copan Vi-Pak Amies Agar Gel collection and transport swabs, and BBL Port-A-Cul) for maintenance of anaerobic and fastidious aerobic organisms.

Of utmost importance in evaluations of clinical samples for infectious agents is proper specimen transport to the clinical laboratory. In the present study we compared three transport systems (the new Starplex StarSwab II, the new Copan Vi-Pak Amies Agar Gel collection and transport swabs, and the BBL Port-A-Cul) for survival of anaerobic and fastidious aerobic bacteria. The new Copan Vi-Pak system has been modified by nitrogen gas flushing to keep an ideal low E(h) condition and to prevent oxidation of the transport medium. The Copan Vi-Pak system outperformed the other two swabs evaluated by maintaining the viabilities of both anaerobic and fastidious aerobic bacteria for 24 h for the majority of the organisms evaluated. This time period should be sufficient for transport of specimens to the clinical microbiology laboratory without compromising organism recovery.

Laboratory diagnosis of atypical pneumonia.

"Atypical pneumonia" is a term loosely applied to lower respiratory tract infections that are not characterized by signs and symptoms of lobar consolidation. This description can apply to disease caused by a variety of bacterial, viral and even protozoan organisms. In reality, differentiation as to etiology of pneumonia cannot be distinguished on the basis of clinical presentation. This review will discuss the epidemiology, clinical manifestations, and laboratory diagnosis of Mycoplasma pneumoniae, Chlamydia sp., Legionella sp., Bordetella pertussis, and Coxiella bumetii, the most common agents associated with atypical pneumonia. Unfortunately, because many of these pathogens are intracellular, culture systems are either not available or the techniques employed are costly, time-consuming or unsafe. Until molecular techniques are standardized and widely available, diagnosis will depend upon serologic confirmation. Given the relative importance of these organisms as causes of community acquired pneumonia, current practice guidelines recommend empiric therapy with a macrolide in patients well enough to be treated as an outpatient. However, diagnostic tests should be performed in any patient requiring hospitalization.

Rapid detection of Campylobacter jejuni in stool specimens by an enzyme immunoassay and surveillance for Campylobacter upsaliensis in the greater Salt Lake City area.

The Alexon-Trend, Inc. (Ramsey, Minn.), ProSpecT Campylobacter microplate assay was compared with culture on a Campy-CVA plate (Remel, Lenexa, Kans.) and blood-free campylobacter agar with cefoperazone (20 microg/ml), amphotericin B (10 microg/ml), and teicoplanin (4 microg/ml) (CAT medium; Oxoid Limited, Hampshire, England) with 631 patient stool samples. The CAT medium was used to isolate Campylobacter upsaliensis. The enzyme immunoassay (EIA) had a sensitivity and a specificity of 89 and 99%, respectively, and the positive and negative predictive values were 80 and 99%, respectively. Even though we extensively looked for C. upsaliensis in stool samples from patients from the greater Salt Lake City area, we did not isolate this species during the study period. The overall excellent specificity of the EIA allows rapid detection and treatment of positive patients; however, a negative result should be confirmed by culture when clinical suspicion is high.

Evaluation of BioStar FLU OIA assay for rapid detection of influenza A and B viruses in respiratory specimens.

BACKGROUND: Demand for the rapid diagnosis of influenza infections has increased with the advent of the availability of neuraminidase antiviral therapy for influenza A and B. Several rapid assays that detect both influenza A and B are now available. OBJECTIVES: In this study we compared the performance of the BioStar FLU OIA assay to Bartels Viral Respiratory Screening and Identification Kit (Bartels Inc., Issaquah, WA), and cell culture. STUDY DESIGN: A total of 145 patient specimens for influenza virus detection submitted in either viral transport medium or in sterile containers were evaluated by the three methods. Specimen types included nasal washings, nasal swabs, sputum, throat swabs, and bronchial alveolar lavage (BAL) fluids. RESULTS: Fifty six positive specimens were identified based on culture and/or DFA. Of these, 30 specimens were positive by the OIA assay for an overall sensitivity of 54%. The OIA assay detected 48% (n = 21) of the 44 culture positive specimens and 81% (n = 29) of the 36 DFA positive specimens. Eighty six of the 89 culture/DFA negative samples were negative by the OIA assay (97% specificity). Analysis of the OIA assay sensitivity from samples submitted in M4 transport medium or in sterile containers revealed that M4 transport medium does not reduce the sensitivity of the OIA assay. Fifteen of the 27 positive samples submitted in M4 transport medium were positive by the OIA assay (56% sensitivity) compared to 15 of 29 positive samples transported in sterile containers (52% sensitivity). Twelve specimens were either culture and/or DFA positive for viruses other than influenza, but negative by the OIA assay, suggesting that there was no cross reactivity of the OIA assay with the other virus types recovered in this study. CONCLUSIONS: The overall excellent specificity of the BioStar FLU OIA allows for treatment of positive patients for influenza, however, a negative result should be confirmed by DFA and culture.

Poliovirus mutants at histidine 195 of VP2 do not cleave VP0 into VP2 and VP4.

The final stage of poliovirus assembly is characterized by a cleavage of the capsid precursor protein VP0 into VP2 and VP4. This cleavage is thought to be autocatalytic and dependent on RNA encapsidation. Analysis of the poliovirus empty capsid structure has led to a mechanistic model for VP0 cleavage involving a conserved histidine residue that is present in the surrounding environment of the VP0 cleavage site. Histidine 195 of VP2 (2195H) is hypothesized to activate local water molecules, thus initiating a nucleophilic attack at the scissile bond. To test this hypothesis, 2195H mutants were constructed and their phenotypes were characterized. Consistent with the requirement of VP0 cleavage for poliovirus infectivity, all 2195H mutants were nonviable upon introduction of the mutant genomes into HeLa cells. Replacement of 2195H with threonine or arginine resulted in the assembly of a highly unstable 150S virus particle. Further analyses showed that these particles contain genomic RNA and uncleaved VP0, criteria associated with the provirion assembly intermediate. These data support the involvement of 2195H in mediating VP0 cleavage during the final stages of virus assembly.