Fourteen isolations of West Nile (WN) virus were obtained from four mosquito species  in statewide surveillance conducted from June through October 2000. Most isolates were obtained from mosquitoes collected in densely populated residential locales in Fairfield and New Haven counties, where the highest rates of dead crow sightings were reported and where WN virus was detected in 1999. Minimum field infection rates per 1,000 mosquitoes ranged from 0.5 to 1.8 (county based) and from 1.3 to 76.9 (site specific). Cx. restuans appears to be important in initiating WN virus transmission among birds in early summer; Cx. pipiens appears to play a greater role in amplifying virus later in the season. Cs. melanura could be important in the circulation of WN virus among birds in sylvan environments; Cx. salinarius is a suspected vector of WN virus to humans and horses.


We evaluated West Nile (WN) virus seroprevalence in healthy horses, dogs, and cats in New York City after an outbreak of human WN virus encephalitis in 1999. Two (3%) of 73 horses, 10 (5%) of 189 dogs, and none of 12 cats tested positive for WN virus-neutralizing antibodies. Domestic mammals should be evaluated as sentinels for local WN virus activity and predictors of the infection in humans.


Human norovirus (NoV) causes more than 80% of nonbacterial gastroenteritis in Europe and the United States. NoV transmission via contaminated surfaces may be significant for the spread of viruses. Therefore, measures for prevention and control, such as surface disinfection, are necessary to interrupt the dissemination of human NoV. Murine norovirus (MNV) as a surrogate for human NoV was used to study the efficacy of active ingredients of chemical disinfectants for virus inactivation on inanimate surfaces.The inactivating properties of different chemical biocides were tested in a quantitative carrier test with stainless steel discs without mechanical action. Vacuum-dried MNV was exposed to different concentrations of alcohols, peracetic acid (PAA) or glutaraldehyde (GDA) for 5 minutes exposure time. Detection of residual virus was determined by endpoint-titration on RAW 264.7 cells.10 within 5 minutes exposure time, whereas 2-propanol showed a reduced effectiveness even at 60% (v/v). Furthermore, there were no significant differences in virus reduction whatever interfering substances were used. When testing with ethanol, 1- and 2-propanol, results under clean conditions were nearly the same as in the presence of dirty conditions (0.3% BSA plus 0.3% erythrocytes).PAA [1000 ppm], GDA [2500 ppm], ethanol [50% (v/v)] and 1-propanol [30% (v/v)] were able to inactivate MNV under clean conditions (0.03% BSA) on the carriers by ≥ 4 logProducts based upon PAA, GDA, ethanol and 1-propanol should be used for NoV inactivation on inanimate surfaces. Our data provide valuable information for the development of strategies to control NoV transmission via surfaces. Norovirus and belong to the family Caliciviridae possessing a single-stranded genome without an envelope. NoV is responsible for more than 80% of nonbacterial gastroenteritis in Europe and the United States [Human noroviruses (NoVs) are members of the genus d States ,2. The mIn young children, a long-term shedding for more than one month was observed in 22.6% of those with adequate follow-up . This stVesivirus and it is known to be a respiratory pathogen. Therefore, survival and inactivation characteristics may differ from those of human NoVs, which are transmitted by the fecal-oral route.Due to the lack of a cell culture system for important nosocomial pathogens such as human NoVs, it is necessary to use a surrogate virus which can easily be cultivated in cell culture. In the past, the most suitable surrogate for human NoVs when studying the virucidal activity of chemical disinfectants in suspension and on carriers was the feline calicivirus (FCV) -7. WithiNorovirus and passes through the gastrointestinal tract. Because of this, MNV seems to be a more suitable surrogate for human NoV than FCV [Recently, propagation of the murine norovirus (MNV) in dendritic cells and macrophages was achieved . Like huthan FCV ,10.This study presents the results of activities of different biocides against the MNV dried on stainless steel to simulate practical conditions. These data should facilitate our understanding of inactivation of human NoV by surface disinfectants.2 until 70–95% of the cells showed a cytopathic effect (after 1–2 days). The cells were frozen and thawed twice, followed by centrifugation at 1600 g for 10 minutes. The supernatant was aliquoted as test virus suspension and stored at -80°C.Test virus suspension was prepared by infecting RAW 264.7 cells (ATCC TIB-71) with MNV  by using a multiplicity of infection of 1. Following 2 hours of adsorption at 37°C, the inoculum was replaced by Dulbecco's modified Eagle's medium  with 3% fetal calf serum . The tissue culture flask was incubated at 37°C and 5% CO3, pH 7.0) prepared according to EN 14476:2007-02 and used within 2 hours [Dilutions of peracetic acid (PAA), glutaraldehyde (GDA) and the different kinds of alcohol  were prepared immediately before the inactivation experiments with hard water (300 ppm CaCO 2 hours .The virus inoculum was prepared by mixing the test virus suspension with the interfering substances according to EN 14476:2007-02 :a) Nine volumes of test virus suspension were mixed with one volume of 0.3% w/v of bovine serum albumin (clean conditions)b) Nine volumes of test virus suspension were mixed with one volume of 3% w/v of bovine serum albumin plus 3% v/v washed sheep erythrocytes (dirty conditions).®90-solution  for 1 h. Afterwards the discs were rinsed off twice with freshly destilled water for 10 sec, ensuring that the carriers did not dry to any extent, and were then placed in 70% ethanol (v/v) for 15 min. Finally, the carriers were dried by evaporation in sterile petri dishes in a biological safety cabinet.For cleaning, stainless steel discs  were incubated in a 5% (v/v) Decon50 μl of the virus inoculum were pipetted in the middle of each pre-treated carrier and dried in a desiccator with a vacuum of 700–800 mbar for about 30 minutes to reach a constant humidity in all experiments. After drying, the virus-contaminated discs were transferred into 25 ml plastic vial holders , which were previously filled with 0.5 g of glass beads  to increase virus recovery by mechanical abrasion. 100 μl of the test biocides were then pipetted on the dried virus inoculum and incubated for 5 minutes. Control carriers received 100 μl of hard water instead of the chemical substance. In order to stop the activity of the test substance, 900 μl of culture medium were added immediately at the end of the exposure time. The vials were vortexed directly for 1 min to recover the residual virus before the eluate was diluted to determine viral infectivity. Each test was carried out with 3 replicates (carriers) per concentration of the substance and virus control respectively, with a minimum of two independent experiments.10 reduction.Infectivity was determined by endpoint dilution titration onto RAW 264.7 cells. At the end of the exposure time, aliquots of the mixture were taken and diluted immediately. 100 μl of each dilution were placed in eight wells of a sterile 96-well microtiter plate with the permissive RAW 264.7 cells. Plates were incubated for 4 to 5 days and infectivity was analysed by virus-induced cytopathic effect. Virus titres were determined by the method of Spearman and Kärber. Titre reduction was calculated as the difference between the virus titre of the water control (5 min) and the titre of the biocides after 5 min of exposure times and is presented as log10 reduction in this study (inactivation of 99.99%).The criterion used for virucidal activity of the biocide was a four log10. Testing GDA the concentration had to be increased up to 2500 ppm to achieve a 4 log10 reduction in virus titre  2-propanol only reduced the viral titre by 3.02 loge Figure , 50–55% 0 Figure . The mos0 Figure .To compare the influence of soil load, we analyzed the three alcohols for their inactivation properties against MNV under clean and dirty conditions. Reduction factors achieved with the corresponding alcohols in concentrations of 40% (v/v) and 60% (v/v) under clean conditions were comparable to those measured under dirty conditions Figure .Norovirus infections spread rapidly particularly in senior residences, hospitals, hotels, schools and even cruise ships. Apparently, contaminated surfaces may play an important role in the spread of human NoV -15. In aMany surface cleaners in the household are based on 0.5% hypochlorite. It was shown by reverse transcriptase polymerase chain reaction (RT-PCR) for human NoVs that the cleaning of fecally contaminated surfaces with a cloth soaked in detergents or treatment with 0.5% hypochlorite alone failed to eliminate human NoVs. The virus could still be detected on up to 28% of the surfaces by RT-PCR. Only wiping the surface with a cloth soaked in detergents followed by applying a combined hypochlorite/detergent treatment was successful to achieve a sufficient efficacy against human NoVs .Since there is only limited knowledge about human NoV inactivation on surfaces, it was the aim of this study to evaluate the virucidal properties of some often-used chemical ingredients of surface disinfectants in a standardized test procedure. The importance of carrier-based methods to test efficacy of disinfectants against vertebrate viruses has been stressed .Meanwhile, carrier test methods including a ring trial launched under the initiative of the Organisation for Economic Cooperation and Development have been published -21. In oThe absence of a cell culture assay for human NoV makes it necessary to introduce a reliable surrogate virus when studying the environmental persistence and efficacy of chemical disinfectants. Because FCV can be propagated in cell culture, it has been extensively studied in inactivation studies in the past ,23. In c10 (data not shown in table). In contrast, the poliovirus titre decreased by about 3 log10 during the drying process [In this study, MNV showed a remarkable stability while drying. The difference in the virus titre before and after drying on the carrier did not exceed 0.35 log process .In general, non-enveloped viruses can persist for approximately up to two months on inanimate surfaces . For exa10 reduction was observed [MNV can persist in various environmental conditions. After 40 days at -20°C and 4°C only a < 2 logobserved . This stRecently, the stability of MNV mixed with artificial feces on stainless steel coupons was studied . In this10 within 5 minutes of exposure time. When testing PAA a concentration of 0.1% (1000 ppm) and when testing GDA a concentration of 0.25% (2500 ppm) was needed to inactivate 99.99% of the viruses. These results were confirmed by the ability of two commercial products based upon GDA and PAA to inactivate MNV [In this study we show that PAA and GDA were able to reduce the titre of MNV under clean conditions about more than 4 logvate MNV .In tests with other viruses simulating practical conditions, 2.0% GDA was able to inactivate HIV, rotavirus and HAV on carriers within one minute -33. Addi10 within a contact time of 10 minutes [Generally, besides PAA and GDA, chlorine-based products are often recommended to inactivate non-enveloped viruses on contaminated inanimate surfaces. Testing nonporous and porous surfaces, 20 to 200 ppm of hypochlorous acid solution reduced the titres of human NoV and MNV by 3 log minutes .10 reduction. These results are in contrast to data with FCV. Here, 2-propanol inactivating 99% within one minute at 40 to 60% was found to be more effective than ethanol (6).In further experiments we analysed the virucidal properties of ethanol, 1-propanol and 2-propanol against MNV in the quantitative carrier test under clean conditions with concentrations from 10 to 60% (v/v). Since results from the suspension test with the MNV where ethanol in a concentration of 60% (v/v) to 80% (v/v) was more effective than 1-propanol followed by 2-propanol (unpublished), we expected ethanol to be the most effective virucidal agent, followed by 1- and 2-propanol. Surprisingly, 1-propanol was the most effective alcohol to inactivate MNV on the carrier under clean conditions. Interestingly, a concentration of only 30% (v/v) 1-propanol was sufficient to inactivate 99.99% of the virus. In comparison, the two other alcohols were completely ineffective at this concentration. Ethanol achieved a similar effectiveness only at 50–55% (v/v), while 2-propanol at a concentration of 60% (v/v) did not reach a 4 log10 reduction detectable between clean and dirty conditions. All three alcohols tested showed similar reduction factors at 40% (v/v) and 60% (v/v) with 0.03% BSA as well as with 0.3% BSA plus 0.3% erythrocytes. At the tested concentration of 40% (v/v) 1-propanol was highly active against MNV after an exposure time of 5 minutes by reaching a reduction of 6 log10, whereas ethanol and 2-propanol could not inactivate the virus sufficiently. The effectiveness of ethanol and 2-propanol could be enhanced by increasing the concentration of alcohol. At an alcohol concentration of 60% (v/v) ethanol reached an efficacy comparable to 1-propanol, whereas 2-propanol was not capable of reducing the virus titre by more than 3 log10 at the same concentration.Approaching practical conditions, the soil load was increased from 0.03% BSA (clean conditions) to 0.3% BSA plus 0.3% sheep erythrocytes (dirty conditions). Previous results of suspension tests or carrier assays had shown that a higher organic load resulted in a decreased effectiveness of the biocide -37. Inte10 within 5 minutes.In summary, our data indicate that 1-propanol was the most effective alcohol to inactivate MNV on stainless steel discs within 5 minutes of contact time. Independent of the amount of soil load, even a concentration of 30% (v/v) 1-propanol had the capability to decrease the virus titre by ≥ 4 logIn further studies, it would be interesting to compare the inactivating properties of these biocides against MNV on other carriers often used in hospitals and other medical settings.To interrupt the transmission of human NoV it is necessary to use highly effective surface disinfectants tested under practical conditions. Our results show that MNV as a surrogate for human NoV is inactivated under clean conditions by PAA and GDA as well as by the two alcohols ethanol and 1-propanol on stainless steel discs after 5 minutes of exposure time, whereas 2-propanol only achieved insufficient effectiveness. The effective concentrations of the chemical biocides examined provide an informative basis when evaluating these substances as possible active ingredients of surface disinfectants with short contact times.Dr. P. Goroncy-Bermes is employed by Schülke & Mayr, Norderstedt, GermanyAll authors contributed to the conception and analysis of data. TM carried out the experiments. All authors are involved in drafting the manuscript. All authors have read and approved the final version of the manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2334/9/107/prepub


Dicistroviridae  includes species that infect insects and other arthropods. These viruses have a linear positive-sense ssRNA genome of ~8-10 kb, which contains two long ORFs. The 5' ORF encodes the nonstructural polyprotein while the 3' ORF encodes the structural polyprotein. The dicistroviruses are noteworthy for the intergenic Internal Ribosome Entry Site (IGR-IRES) that mediates efficient translation initation on the 3' ORF without the requirement for initiator Met-tRNA. Acute bee paralysis virus, Israel acute paralysis virus of bees and Kashmir bee virus form a distinct subgroup within the Dicistroviridae family. In this brief report, we describe the bioinformatic discovery of a new, apparently coding, ORF in these viruses. The ORF overlaps the 5' end of the structural polyprotein coding sequence in the +1 reading frame. We also identify a potential 14-18 bp RNA stem-loop structure 5'-adjacent to the IGR-IRES. We discuss potential translation initiation mechanisms for the novel ORF in the context of the IGR-IRES and 5'-adjacent stem-loop.The family  Dicistroviridae includes a number of insect- and arthropod-infecting species such as Cricket paralysis virus, Black queen cell virus, Plautia stali intestine virus and Taura syndrome virus. The species Acute bee paralysis virus (ABPV), Israel acute paralysis virus of bees (IAPV) and Kashmir bee virus (KBV) - which have been associated with Colony Collapse Disorder of honeybees - form a tight subclade within the family  or any of the usual initiation factors  was apparent at the 5' end of CDS2  a portion of ribosomes, or rather 40S ribosome subunits, binding to the IGR-IRES somehow start scanning, and normal AUG-initiation takes place at a conserved tandem pair of +1 frame AUG codons ~35 codons downstream  a portion of ribosomes initiate at more-or-less the normal IGR-IRES mediated non-Met-tRNAm Figure  or, in sThe synonymous site conservation plot peaks around the tandem +1 frame AUG codons Figure , fallingIf ORFX initiation occurs at the normal IGR-IRES initiation site but in the +1 frame then translation of ORFX would result in an 11.2 kDa, 93 amino acid product in KBV, and 92 and 94 amino acid products in ABPV and IAPV respectively. If, however, initiation takes place at the downstream tandem AUG codons, then translation of ORFX would result in a 7.1 kDa, 60 amino acid product in all three species. Within the longer (i.e. 92-94 amino acid) potential ORFX product, there are 61 residues that are completely conserved between the KBV, ABPV and IAPV GenBank RefSeqs. In the region of the structural polyprotein that is encoded by the portion of the CDS2 sequence that ORFX overlaps, there are 66 completely conserved residues. Thus the putative ORFX product is apparently subject to slightly weaker functional constraints than the 'corresponding' portion of the structural polyprotein.NC_006559]; [NC_003005]; ; ).FJ229495] - with partial coverage of the ORFX region contained an ORFX-frame premature termination codon [PTC] that truncates ORFX by 33 codons. However, apart from the potential for sequencing errors, PTCs in a small number of isolates are not unusual for short overlapping genes, which tend to have non-essential 'secondary' functions, and we do not believe that this ORFX-defective partial sequence necessarily precludes the presence of a functional ORFX in SINV-1.)SINV-1 clusters with the bee paralysis viruses in the phylogenetic tree Figure .  potential RNA hairpin structure immediately 5'-adjacent to, but not overlapping, the IGR-IRES in the bee paralysis viruses Figure . In the i [Recent results suggest that under certain circumstances (namely the presence of an initiator tRNA species that recognizes the P-site codon) the IGR-IRES can, at some level, mediate initiation at the P-site (presumably in competition with A-site initiation) . The codi .Other dicistroviruses lack a long overlapping ORF at this genomic location and lack the corresponding extended region of synonymous site conservation (data not shown). At least some other dicistroviruses do exhibit some degree of heightened synonymous site conservation at the very 5' end of CDS2, but the 3' extent of these regions appears to be much more limited than in the bee paralysis viruses . In facOverlapping genes are difficult to identify and are often overlooked. However, it is important to be aware of such genes as early as possible in order to avoid confusion (otherwise functions of the overlapping gene may be wrongly ascribed to the gene they overlap), and also so that the functions of the overlapping gene may be investigated in their own right. Although overlapping the structural polyprotein, there is no reason to suspect that ORFX encodes a structural protein - indeed the limited phylogenetic distribution of ORFX suggests that it does not. We are currently investigating the translation mechanism for the putative ORFX and how it relates to the IGR-IRES and the potential upstream hairpin structure.Note: during the preparation of this manuscript, the positive coding potential of ORFX was also predicted by Ref.  (who namThe authors declare that they have no competing interests.AEF carried out the bioinformatic analysis and wrote the manuscript. All authors edited and approved the final manuscript.


Eradication strategies must consider all cellular sources of virus. During the course of infection, HIV-1 can evolve to acquire new cell tropism. We have examined virus in blood and cerebral spinal fluid to identify virus capable of infecting macrophages.Viral env genes were amplified by PCR using limiting template dilution. The amplicons were sequenced and a subset was cloned into an expression vector. The cloned env genes were used to create pseudotyped virus that was then tested for entry into Affinofile cells expressing high and low levels of CD4.We examined virus in blood and CSF in eight subjects with advanced HIV disease and with neurocognitive involvement. All eight subjects had highly compartmentalized virus in comparing blood and CSF. In half of the subjects, the virus decayed rapidly in blood and CSF when therapy was initiated, and all env genes tested could infect cells only when there were high levels of CD4, both features indicative of infection of activated T cells in both the blood and central nervous system (CNS). In the other half of the subjects, virus decayed slowly in the CSF, but rapidly in the blood with the initiation of therapy, and the virus in the CSF could infect cells with low levels of CD4, indicative of the presence of macrophage-tropic virus in the CNS. However, the majority of the virus in the blood retained the requirement for high levels of CD4 for infection.Macrophage-tropic viruses evolve late in disease and are compartmentalized in the CNS and perhaps elsewhere. They are produced from cells with a long half-life compared with activated T cells. Finally, they are poorly represented in the blood, even when they predominate in the CNS.


To determine if eastern North American Ixodes dammini, like related ticks in Eurasia, maintain tick-borne encephalitis group viruses, we analyzed ticks collected from sites where the agent of Lyme disease is zoonotic. Two viral isolates were obtained by inoculating mice with homogenates from tick salivary glands. The virus, which was described by reverse transcriptase polymerase chain reaction and direct sequencing of the amplification products, was similar to, but distinct from, Powassan virus and is provisionally named "deer tick virus." Enzootic tick-borne encephalitis group viruses accompany the agents of Lyme disease, babesiosis, and granulocytic ehrlichiosis in a Holarctic assemblage of emergent deer tick pathogens.


A traditional single-stage reverse transcription-polymerase chain reaction (RT-PCR) procedure is effective in determining West Nile (WN) virus in avian tissue and infected cell cultures. However, the procedure lacks the sensitivity to detect WN virus in equine tissue. We describe an RT-nested PCR (RT-nPCR) procedure that identifies the North American strain of WN virus directly in equine and avian tissues.


To the Editor:Dengue virus belongs to the genus Flavivirus, family Flaviviridae. It has 4 serotypes: dengue virus type 1 (DENV-1), dengue virus type 2 (DENV-2), dengue virus type 3 (DENV-3), and dengue virus type 4 (DENV-4). Dengue virus is maintained in a cycle between humans and Aedes aegypti, domestic day-biting mosquitoes. Dengue virus induces clinical illness, which ranges from a nonspecific viral syndrome (dengue fever [DF]) to severe and fatal hemorrhagic disease (dengue hemorrhagic fever [DHF]). DF/DHF occurs primarily in tropical and subtropical areas of the world. Domestic dengue virus infection occurs in >100 countries; >2.5 billion persons live in these areas. Approximately 100 million cases of DF, 500,000 cases of DHF, and several thousand deaths occur annually worldwide (3]) consistent with the World Health Organization (WHO) definition of DHF. Ascites and plural effusion developed in 2 patients. Blood specimens were collected from all patients at the time of admission to the local hospitals. Particle agglutination (PA) assay   . Each ofDF/DHF have been considered to be a possible public health threat to Nepal because DF/DHF epidemics have occurred recently in India and Pakistan, which reported several thousand cases and >100 deaths (Aedes mosquito is known to persist in this region. The emerging DENV-2 is likely to have been introduced into Nepal from India.The emergence occurred in the lowland Terai belt region, which borders the state of Bihar, India. The


New sequencing technologies are rapidly emerging. Many laboratories are simultaneously working with the traditional Sanger ESTs and experimenting with ESTs generated by the 454 Life Science sequencers. Though Sanger ESTs have been used to generate contigs for many years, no program takes full advantage of the 5' and 3' mate-pair information, hence, many tentative transcripts are assembled into two separate contigs. The new 454 technology has the benefit of high-throughput expression profiling, but introduces time and space problems for assembling large contigs.The PAVE (Program for Assembling and Viewing ESTs) assembler takes advantage of the 5' and 3' mate-pair information by requiring that the mate-pairs be assembled into the same contig and joined by n's if the two sub-contigs do not overlap. It handles the depth of 454 data sets by "burying" similar ESTs during assembly, which retains the expression level information while circumventing time and space problems. PAVE uses MegaBLAST for the clustering step and CAP3 for assembly, however it assembles incrementally to enforce the mate-pair constraint, bury ESTs, and reduce incorrect joins and splits. The PAVE data management system uses a MySQL database to store multiple libraries of ESTs along with their metadata; the management system allows multiple assemblies with variations on libraries and parameters. Analysis routines provide standard annotation for the contigs including a measure of differentially expressed genes across the libraries. A Java viewer program is provided for display and analysis of the results. Our results clearly show the benefit of using the PAVE assembler to explicitly use mate-pair information and bury ESTs for large contigs.The PAVE assembler provides a software package for assembling Sanger and/or 454 ESTs. The assembly software, data management software, Java viewer and user's guide are freely available. ESTs have been prevalent in genomic research since the first large scale EST project in 1991 -4. In JaBesides the 454 sequencer, the following are also next-generation sequencers that generate high-throughput short reads: Illumina Genome Analyzer ,7, and HMany current EST projects have generated 454 data and either used traditional EST assembly approaches (e.g. ) or aligThe ESTs generated by Sanger versus the 454 sequencer differ in number and length. The current Sanger ESTs have an average length of around 650 good bases, but the number of ESTs that are sequenced is generally low. The cDNA sample must first be cloned into a vector  to produce a cDNA library and then individual clones are isolated from the library and sequenced, which results in a few thousand clones being sequenced. For example, most maize libraries in GenBank have between 1000 and 10,000 ESTs. The 454 GS FLX sequencer can generate over 200,000 good ESTs per project, but at an average length of only 250 trimmed bases. The new 454 GS FLX Titanium is capable of generating over a million reads of 400 bases with reduced error [Sanger sequencing can produce mate-pairs where it is known which ESTs are mates based on their name. If the clone is full length, the 5' end will start at the beginning of the transcript, otherwise it can start anywhere within the original mRNA sequence. It is now relatively inexpensive to generate both the 3' and 5' reads of a clone, as the library only needs to be prepped once. To date no software exists that takes full advantage of mate-pair information in order to produce better contigs. CAP3  uses matThe advent of 454 sequences presents new challenges to assembly. The increased depth of the 454 data sets can cause CAP3 and other assembly programs to run out of memory. Moreover, assembling large contigs (e.g. > 1000 ESTs) is time-consuming. To address both problems, ESTs contained in another may be removed, such as performed by PlantGDB ,24. PAVEThere are quite a few packages for the pipeline processing that EST data requires  returns true if the sequences sx and sy have a minimum overlap of m, a minimum identity of d%, and a maximum overhang of h. The small amount of overhang allowed by h is necessary on the end of the overlapping portion to allow for poorly trimmed sequence. The parameters mi, di and hi are set by the user in a configuration file (there are three or more sets as explained below). The OK_CTG(rulen) function analyzes the results of CAP3 to determine whether to accept or reject the contig. There are three sets of rules that have been designed by us based on inspection of contigs using the jPAVE graphical interface, and will be discussed in the section on heuristics.Guiding the system are the OK_OLAP and OK_CTG functions, which contain rules for acceptance or rejection of MegaBLAST and CAP3 output, respectively. OK_OLAP with every other mate-pair in the clique. Each clique is assembled and if the CAP3 results pass OK_CTG(rule1), it is retained as a seed contig. (ii) Compute singleton cliques where each singleton must pass OK_OLAP with every other singleton in the clique. Each clique is assembled and if the CAP3 results pass the OK_CTG(rule2) function, it is retained as a seed contig. (iii) Each remaining mate-pair is made into a contig, where the mates are joined if they pass OK_OLAP. (iv) Each remaining singleton is made into a contig. The purpose of assembling cliques is that the existence of a set of mate-pairs (or singletons) that all mutually overlap creates a strong contig and it reduces the time of the next step. This step produces the initial contig consensus sequences (CCS).The clique step creates seed contigs from the file of EST-against-EST comparison results, as follows: (i) Compute mate-pair cliques where each mate-pair must pass OK_OLAP, and the edge is weighted by the bit-score. The nodes of a given TC are incrementally assembled together where the order is based on bit score and the number of ESTs. If a pair assembles correctly according to OK_CTG(rule3), the two nodes are collapsed into one. The TCs are assembled in parallel, i.e. each TC is assigned to a separate compute node for incremental assembly. The parallelism is implemented with the standard Unix fork function to take advantage of multiple processors.The TC step computes the set of TCs from the file of CCS-against-CCS comparison results. Each TC is a connected graph where each node is a contig, an edge exists between each two contigs that pass OK_OLAP, i = 3 to n+3, where the n corresponding parameters are provided in the configuration file. For Sanger ESTs, we typically use two iterations, which merges over-split contigs; this approach of using a second assembly has been used by others, e.g. PlantGDB [The TC step is executed PlantGDB . For 454Two sub-contigs are not joined by n's until after assembly, at which point the orientation of the two sub-contigs needs to be computed. This is computed by a majority rule on the orientation and forward/reverse information of the ESTs.The OK_CTG function evaluates the output of CAP3 and accepts it if it results in one contig and rejects it if it results in more than two contigs (including singletons). If it results in two contigs, there must be more than one mate-pair bridging the two sub-contigs and all orientations must be consistent. These two rules are relaxed on the last iteration of assembling TCs in order to allow for the more common exceptions: (i) Two small sub-contigs may be bridged by one mate-pair. (ii) The sub-contigs may have mixed orientation, for example, both ends of the last mate-pair shown in Figure A chimeric clone may have one half of the mate-pair belong in one contig and the other half in another contig. By forcing mate-pairs to be in contigs together, a chimeric clone will generally be in a contig of its own. The advantage is that it reduces chimeric multi-clone contigs resulting from chimeric clones. The disadvantage is that it may cause redundant contigs, as one half of the single mate-pair contig may align perfectly to another contig.To substantially decrease usage of computer time and space, ESTs are buried both before and during the assembly process, i.e. if EST B is contained in EST A, it can be "buried" in the "parent" EST A and removed from assembly. ESTs may be buried prior to assembly when one EST is contained in another based on the BLAST coordinates; the amount of mismatches allowed is a user-supplied parameter. During assembly, additional ESTs are buried if one is contained within another based on CAP3 coordinates, even though there may be mismatches between the buried and parent ESTs. For mate-pairs, both mates must be contained in the same parent clone in order to be buried. Buried ESTs are recorded in a file and when the PAVE algorithm completes and the contigs are loaded into the database, the buried clones are assigned the same contig as the parent.The algorithm for burying ESTs  ensures that the remaining unburied ESTs are sufficient to provide a good coverage of the contig. Briefly, the algorithm builds a containment tree and then performs a breadth-first traversal of the tree, retaining all top-level ESTs plus a number of ESTs from the next-highest levels based on configuration parameters. The breadth-first search serves to distribute the retained ESTs evenly across the contig. After retaining a sufficient number for good coverage, the rest of the ESTs in the tree are buried. For burial during assembly, the unburied ESTs are re-CAP'ed, and if they fail to assemble together, the attempt to bury the clones is rejected. Since this re-CAP takes time, the burial is only attempted if there are at least Y (default 100) ESTs in a contig and at least × (default 25) ESTs can be buried.A post-processing routine reassembles the contigs that have buried ESTs using all ESTs in the contig, primarily in order to provide accurate coordinates for the buried clones. The re-CAP occasionally fails due to too many ESTs or due to the mismatches from the CAP3 buried ESTs; in these cases, the ESTs are positioned under their parent clone and shown in red.As we have multiple PAVE projects, each with many EST libraries, it was advantageous to create an organized data management system as part of PAVE. This is done with a set of configuration files and a rigid directory structure, where there is a directory for libraries and another for assembly projects. By separating the libraries from the assemblies, different projects can be created with overlapping sets of libraries. The package provides four programs: (i) The loadLibrary.pl script loads one or more libraries of ESTs and metadata  into the MySQL database. (ii) The runPAVE.pl script assembles the ESTs and enters the results into the database. (iii) The capBuried.pl script runs CAP3 on all the contigs with buried ESTs in order to assign correct coordinates. (iv) The jPAVE program provides some annotation and an interactive display.After assembling the contigs, the runPAVE.pl script computes the R statistic , which iThe jPAVE program can be run as either as a standalone program or a web applet. The initial window shows all PAVE projects, where any number can be selected for viewing (e.g. for comparing assemblies). As shown in Figure Three sets of results are provided: (1) maize Sanger ESTs with mate-pairs, 2) trichome 454 ESTs, (3) a comparison of PAVE to CAP3 and TGICL with a benchmark set of Sanger mate-pair ESTs. The PAVE parameters used for each assembly are provided in Additional file  trichomeThe maize ESTs were downloaded from GenBank, then filtered to remove the ESTs less than 150 bases in length or submitted earlier than 1999. The resulting 797,619 ESTs had an average length of 585 bases and median of 605 bases. Unfortunately, there is no field in the GenBank record that explicitly identifies a 3' and 5' EST as mate-pairs, but the ESTs from our maize full length cDNA project  are nameThis maize Sanger assembly was executed on a system with two dual-core AMD Opteron 2 GHz processors and 12G RAM. Though the execution time is 6.7 days, very little human time is required to run the assembly. That is, the dataset does not have to be broken up, assembled separately and then merged, as would be necessary with programs that cannot handle datasets of this size. For example, the largest data set we can assemble on this machine with TGICL is 228,000 ESTs.Solanum arcanum  from the Solanum Trichome Project [Our 454 data set is 415,559 ESTs from the stems of  Project . The aveThis trichome 454 assembly was executed on a system with four dual-core Intel Xeon 3.66 GHz processors and 14G RAM, which took almost as long as the much larger maize assembly, even though it was run on a faster machine. This is due to the number of deep contigs as illustrated in Figure We compared PAVE to TGICL and CAP3 for two reasons. First, the majority of EST papers use one of these two programs  was counted as merged if a pair of ESTs in an assembled contig were in different benchmark contigs b1 and b2. A contig pair  was counted as a split contig if a pair of ESTs in a benchmark contig were in different assembled contigs a1 and a2. Table The benchmark ESTs were assembled with the three programs and the results were tested against the benchmark contigs to determine the number of split  and merged  contigs. A contig pair  or de BThe jPAVE assembly viewer provides an excellent way to evaluate contigs. We have assembled a large variety of libraries with different characteristics, and the occasional oddly-aligning contigs often cannot be explained nor anticipated. Having a versatile query and display program such as jPAVE is essential to understanding the wide range of problems that can occur with EST assembly. Moreover, it allows the biologist to diagnose the problems that occur with their specific libraries. For example, by assembling ESTs without the heuristics and then viewing them in jPAVE, the biologist can inspect the amount of chimerism and 3' slippage.pave@agcol.arizona.edu). The software, jPAVE viewer and User's Guide are available from our PAVE website [The PAVE system is created for large projects with multiple libraries. This allows comparing across libraries for differentially expressed genes, which will become more relevant with the large number of ESTs generated by the next-generation sequencing machines. The PAVE system also has a web interface CS conceived the study and wrote the majority of the paper, with aid from all the other authors. EJ and MB provided most of the development work with aid from CS and AD. AD prepared the maize and trichome data sets.List of EST papers. A partial list of EST papers published since 1999, with sections: 1. EST assembly software. 2. EST pre-processing, pipeline and viewing software. 3. EST annotation. 4. EST analysis for one for one or more libraries. 5. Related papers. 6. Typical references in EST papers.Click here for fileParameters and log files. The parameters and content the of log files for the maize, trichomes and benchmark assemblies.Click here for fileBenchmark contigs. A description of the steps and parameters used to create the benchmark contigs.Click here for file


All infectious disease oriented clinical diagnostic assays in use today focus on detecting the presence of a single, well defined target agent or a set of agents. In recent years, microarray-based diagnostics have been developed that greatly facilitate the highly parallel detection of multiple microbes that may be present in a given clinical specimen. While several algorithms have been described for interpretation of diagnostic microarrays, none of the existing approaches is capable of incorporating training data generated from positive control samples to improve performance.Viral Identification using a PRobabilistic algorithm), which uses Bayesian inference to capitalize on empirical training data to optimize detection sensitivity. To illustrate this approach, we have focused on the detection of viruses that cause hemorrhagic fever (HF) using a custom HF-virus microarray. VIPR was used to analyze 110 empirical microarray hybridizations generated from 33 distinct virus species. An accuracy of 94% was achieved as measured by leave-one-out cross validation. ConclusionsTo specifically address this issue we have developed a novel interpretive algorithm, VIPR (VIPR outperformed previously described algorithms for this dataset. The VIPR algorithm has potential to be broadly applicable to clinical diagnostic settings, wherein positive controls are typically readily available for generation of training data. The field of viral diagnostics, which has traditionally followed a "one virus-one assay" paradigm, has been revolutionized by the introduction of diagnostic microarrays -10. It iThe typical goal of diagnostic virology assays is to determine the presence or absence of one or more viruses from a finite, defined list of candidate viruses known to cause the disease in question. In clinical laboratories, samples of each candidate virus to be detected are typically readily available and can be used as positive controls. Our goal was to develop an interpretive algorithm for diagnostic microarrays that could take advantage of the existence of such positive controls to generate a training data set to guide subsequent analyses.Toward this end, we developed a probabilistic algorithm for the purpose of analyzing diagnostic microarrays. This class of algorithms has been applied to numerous problems in biology. For example, hidden Markov models (HMMs) and the more youthful conditional random fields (CRFs) have allowed researchers to make important inferences about sequence structure and function ,19. BayeArenaviridae, Bunyaviridae, Flaviviridae and Filoviridae. A custom microarray was designed to detect all known HF viruses and many of their close relatives. Specimens representing virtually every virus species known to cause HF were procured and hybridized to microarrays for the purpose of validating our algorithm. Furthermore, we compared VIPR's performance to that of the existing interpretive algorithms that are not capable of utilizing training data in this fashion.In this paper, we describe a novel probabilistic algorithm that relies on Bayesian inference for analysis of diagnostic microarrays. To validate this approach, we focused on analysis of the set of viruses known to cause hemorrhagic fever. HF symptoms include severe vascular damage, hemorrhage, high fever, and shock and can frequently lead to death ,21. HF v® 8 × 15 K platform was used and probes were 35, 45 or 60 nucleotides in length. The probes were designed to bind to viral genomes from the four families that contain all viruses known to cause HF: Arenaviridae, Bunyaviridae, Filoviridae, and Flaviviridae. Probes of different lengths were designed to account for different levels of conservation between viral taxa. For example, longer probes were included to represent regions of strong conservation, while shorter probes were included to distinguish closely related virus species in order to increase specificity.14,864 oligonucleotide probes were designed using a taxonomy-based approach as described previously  except t® protocols and was randomly amplified as previously described [® software. In total, 110 hybridizations were performed (102 positive controls + 4 Vero negative controls + 4 C6/36 negative controls). All raw microarray data are available in NCBI GEO (accession GSM534862 through GSM534971). These 110 hybridizations constituted a set of positive and negative controls used for validation, a subset of which was used in training our algorithm.A total of 51 strains of 33 distinct virus species , iNT xis the normalized, transformed value for that intensity. Normalization was performed to account for variation in reagent concentrations or fluorescence across the microarray. Log transformation of the data was desirable for the estimation of normal distributions.For each sample in the training set, a unit-vector normalization was applied as shown, where NT does not appear.Note that in the following calculations, all intensities have been normalized and transformed although the superscript On state for that genome; otherwise, the probe was assigned the Off state. The choice of -30 kcal/mol was based on the observation that this threshold represents the weakest binding reported for long-oligo broad specificity microarrays [On). A total of 101 candidate genomes met these criteria.Candidate genomes to be scored in the VIPR algorithm were limited to all complete genomes in the NCBI virus RefSeq database as of 6/20/2008. The entire set of oligonucleotide probes on the microarray was aligned using BLASTN against each of the RefSeq viral genomes. Theoretical free energies of hybridization were then calculated from the aligned sequences using code included with OligoArraySelector . If the roarrays . A giveni according to Bayes' rule, (2) and (3). i Yand i xrepresent a random variable and an observed intensity, respectively.Posterior probabilities were calculated for each probe e transformed intensities: those corresponding to the On states for a given probe (4), and those corresponding to the Off states (5).Likelihoods for each probe were determined using normal distributions derived from two sets of normalized logOn and Off distributions are derived from the training set where the probe On/Off states are defined by the identity of the virus in each hybridization.The probe-specific On for the candidate virus under consideration, represented as predP(On); (b) the number of candidate viruses that share that probe's On/Off prediction  = 1/4 for that probe). Marginalizing over the possibility of an On or an Off prediction calls for a second invocation of Bayes' rule:Priors were calculated  in a probe-specific manner and were designed to incorporate two calculations derived from the composition of the microarray as well as the composition of the set of candidate viruses under evaluation: (a) the percentage of probes predicted to be x, was calculated for each of n viral genomes. The posteriors included in the product were chosen so as to reflect the expected state of a particular probe for svirus. On states for s virusare indexed from i = 1 to a while Off states are indexed from j = 1 to b as shown in formulas (9) and (10), respectively.Because of the possibility of underflow, all likelihood calculations were made in log space, though they are expressed here in probability space. The likelihood (8) of the observed hybridization vector, P(Off |i Y= i x) so as to estimate a null distribution of scores (n = 100 permutations) against which the actual score for that candidate could be compared. From the 100 null scores for each candidate virus, a mean and standard deviation were calculated. The resulting p-value reflects the percentage of the null distribution that is greater than or equal to the actual score. When assessing the significance of a given candidate, a Bonferroni correction was applied so that 0.05, a generally accepted level of significance, was divided by the total number of candidate viruses (101) i.e. a candidate was considered significant if its p-value was less than 5 × 10-4.To determine the significance of the results obtained, we computed a p-value for each candidate virus by permuting the set of priors for the candidate over the set of likelihoods On. Two hybridizations of Ippy virus to the array were excluded from training because the percentage of probes designed to bind to Ippy virus that evinced a sufficient separation (p < 0.001 by student's t-test) between the On and Off distributions was less than ten percent. For the initial cross-validation, the subset of 108 arrays was divided into a training set consisting of 107 arrays and a validation set consisting of a single array. This was done 108 times, leaving out a different array each time. The two arrays that did not meet the criterion for inclusion in the training set were tested using all 108 selected arrays for training. For each array, the best prediction was determined by sorting significant candidate viruses (p < 5 × 10-4) by p-value and then by likelihood. In the case where no virus was significant, the array was considered negative. Algorithm accuracy was computed using the formula, Accuracy = (TP + TN)/(TP + TN + FP + FN), where TP is the number of true positives, TN is the number of true negatives, FP is the number of false positives, and FN is the total number of false negatives. In the case where the fully sequenced genome of a viral subspecies was not available, an accurate prediction on the species level constituted a true positive. There was also one case where the genome of a subspecies (La Crosse virus) was used as a substitute for a hybridized species  because the complete sequence of California encephalitis virus was not available. These designations of species and subspecies are according to NCBI taxonomy.From the total 110 empirical hybridizations, 108 were chosen as suitable for training on the basis of percentage of well-behaved probes among those predicted to be marg P(On)and marg P(Off)in Equation (2) were replaced with priors that ranged iteratively from 0.1 to 0.9. For each iteration, one prior pair i.e. P(On), P(Off) where P(Off) = 1- P(On) was chosen for all On probes, while a separate pair was chosen for all Off probes. Thus, while the prior pair between the On and the Off probes could differ, the prior pair between any two On probes or between any two Off probes was the same. Hence, the space explored represents successive iterations of independently varying the On prior pair and the Off prior pair with variations made at a step size of 0.1. As before, p-values were calculated to assess the significance of VIPR results, except that 20 permutations were run for each candidate virus instead of 100.In a separate analysis, VIPR's accuracy was assessed over a space of arbitrary priors rather than deliberately specifying priors using Equations (6) and (7). Thus, the marginalized priors Four independent strains of each of the following viruses, Crimean-Congo hemorrhagic fever, Guanarito virus and Omsk hemorrhagic fever virus were cultured in Vero cells. These viruses represent three of the four HF virus families. As with the other viruses in the positive control dataset, these viruses were hybridized in duplicate (3 viruses × 4 strains per virus × 2 hybridizations for each strain = 24 hybridizations). These 24 hybridizations were used to assess the effect that leaving out both replicates of a strain would have on cross-validation. VIPR predictions were made as described for the leave-one-out cross validation except that replicate hybridizations were excluded from training for the subset of 24 arrays. The number of accurate predictions made by VIPR out of the total 24 hybridizations was calculated.-4) was also applied to E-Predict. Background-corrected intensities were loaded into DetectiV [-4) was also applied to DetectiV. Hybridization intensities were inputted to PhyloDetect [i x≥ -60 kcal/mol →1; otherwise → 0). The fnr parameter was set to 0.10. Results were sorted first by likelihood and then by number of present probes to determine the top candidate. A likelihood above the threshold 0.05 constituted a positive prediction. The same formula to calculate accuracy for VIPR was used to calculate accuracies for E-Predict, DetectiV and PhyloDetect.Three algorithms, E-Predict, DetectiV and PhyloDetect, were available for comparison to VIPR. E-Predict  was usedDetectiV  and normloDetect  as binarRNA was purified from cell cultures that were infected with each of the viruses shown in Table http://ibridgenetwork.org/wustl/vipr). VIPR incorporates both sequence data from GenBank as well as empirical array data to classify microarray hybridizations of samples with unknown viral infections  from that probe's Off distribution. To assess VIPR's performance on the 108 selected arrays, we performed leave-one-out cross validation so that the selected arrays were divided into a training set (n = 107) and a validation set (n = 1). The remaining two arrays (not included in the training set) were tested using the entire set of selected arrays (n = 108) for training.For identification of HF viruses, the algorithm was trained using a subset of the total 110 hybridizations. To select a suitable subset for training purposes, we identified 108 hybridizations for which at least 10% of the probes predicted to be An example of VIPR's output for a representative Dengue virus 3 hybridization is shown in Table On versus the pair used for those expected to be Off. Hence, the space explored represents different combinations of prior pairs whose values lie between 0.1 and 0.9 with variations made at step size of 0.1, and with the sum of P(On) and P(Off) defined as 1.0 for each prior pair. 20 permutations were run for each candidate virus to compute p-values. Results are shown in Figure For all Bayesian methods, one question that must be addressed is how to choose appropriate priors. From many possible choices, we selected priors in this study based upon the composition of the probes in the microarray as well as the makeup of the candidate genomes under evaluation. In order to define the dependency of our algorithm's accuracy on the choice of priors, VIPR's accuracy was assessed over a range of possible prior pairs, independently varying the pair used for probes expected to be For the 24 hybridizations representing four strains from each of three species , a second cross-validation was performed in which both replicates corresponding to a particular strain were excluded from training when making VIPR predictions for those arrays. Leaving out both replicates for these particular strains was possible because there remained three other positive control strains of the same species in the training set. This could not be done in the case where only one strain of a species was present among the positive controls because it would render the training set devoid of any representatives of that species. VIPR analysis of the subset of arrays that represent viruses where multiple strains are present in the training set demonstrated robust prediction (24/24 arrays accurately predicted).We compared the performance of VIPR to that of existing algorithms for analyzing diagnostic microarrays. E-Predict , the firDetectiV  is an R-PhyloDetect has previously been applied to viral diagnostic microarrays by increasing its 'false negative rate' parameter . PhyloDeThe inherently parallel nature of DNA microarrays lends itself well to diagnostic applications seeking to simultaneously test for many microbial agents. While many diagnostic microarrays have been described -10 thereOne key feature of a true diagnostic microarray is that the targets to be detected are typically well defined. Thus, specimens infected with these targets should be available for use as positive controls. In this study, we developed a novel interpretive algorithm for analysis of diagnostic microarrays that takes advantage of the existence of positive controls that can serve as a training set. VIPR performed with high accuracy (94%) as measured by leave-one-out cross validation. Since VIPR outperformed E-Predict, DetectiV and PhyloDetect for this dataset, this underscores the utility of using a set of known viruses together with a probabilistic algorithm to diagnose viral disease. Though we have not applied our algorithm to other diseases, we anticipate that this strategy would similarly be preferable to a non-Bayesian approach for diagnosis of other diseases of multiple etiologies whose microbial spectrum is well defined and for which positive and negative control specimens are available.On and Off distributions for probes designed to bind to these viruses reveals that among those viruses that were hybridized to the array, Ippy virus and Kyasanur Forest disease virus exhibited the highest percentage of On probes that displayed no significant difference (p < 0.001) in intensity between the On and Off distributions (94% and 85% respectively). However, since VIPR's accuracy is inherently limited by the performance of the probe set, and the response of the probe set is determined by the identity and abundance of the target microbes, we are unable to distinguish between the possibilities of low-titer virus and misbehaving probes.Only one false positive resulted from the cross-validation, which was a Dengue virus 3 sample being classified as Dengue virus 4. Dengue virus 3 was the second best prediction for this array, with both Dengue virus 4 and Dengue virus 3 achieving a p-value of 0.0. The other five microarrays that were misclassified by VIPR, all of which were called as virus negative, were derived from three virus cultures. None of these samples was accurately classified by E-Predict, DetectiV, or PhyloDetect. Given that these samples evaded accurate classification by all three algorithms, one possibility for the lack or detection of virus in these samples is the samples used as positive controls may have been present in abundance below the sensitivity limit of the microarrays. Another plausible explanation is that all or most of the probes designed to detect these viruses do not behave as predicted. In this case, redesigning the probes for these viruses would be the best way to improve the accuracy of the platform. Comparing the On states for a probe because of the small number of intensities in the training set that correspond to an On state. Hence, one way to improve the accuracy of estimation of these distributions would be to increase the number of positive control arrays in the training set. Depending on the degree of sequence divergence among the known strains of a given virus, it may also be important to represent the known diversity of related strains in the training set. However, we emphasize that even with the limited number of microarray hybridizations performed in this study, 94% accuracy was achieved.Other potential caveats related to our method include a limited ability to estimate the true intensity distribution of e of the intensities was assumed to be normally distributed. Gross violations of this assumption could have pejorative effects on prediction.The choice of prior probabilities could also be problematic in some circumstances. We found that prior estimation based on predicted binding of probes to viral genomes resulted in robust virus prediction. Moreover, accuracy remained fairly stable (between 85% and 97%) over a wide range of prior combinations. Another potential caveat with the VIPR algorithm is that the distribution of the logOne limitation of a leave-one-out cross-validation in our case is that there is a possibility of overfitting due to the presence of replicate hybridizations in the training set. However, an analysis of a subset of arrays that represented several different strains of viruses  demonstrated that removing both replicate hybridizations for a given strain from the training set while retaining those from the other strains resulted in accurate prediction in every case. This subset of viruses represented three of the four families of HF viruses. While this analysis does not completely rule out the possibility of overfitting, it clearly demonstrates that VIPR can make accurate predictions even when replicate arrays are removed from training, as long as hybridizations representing strains from the same species are present. Additionally, VIPR outperformed the other three algorithms for this subset. E-Predict, DetectiV and PhyloDetect accurately classified 14, 16, and 8 of the 24 arrays, respectively.in vivo human infections are available, such would be the desired dataset for training. In the case of HF, however, clinical specimens from human infections are not generally available; therefore, it will be necessary to investigate the use of different kinds of specimens as training data for the probabilistic algorithm. These datasets could include specimens from infected animals or viruses harvested from culture and spiked into human sera.While the results of our study represent a proof of principle using carefully controlled positive and negative controls for validation, it is anticipated that a probabilistic algorithm will be useful in clinical laboratory settings to analyze microarrays like the one described. Testing VIPR using clinical datasets will be the focus of future studies. In the case of diseases for which samples representing On posteriors from different viruses. Equations (11) through (14) extend the single-virus likelihood calculation implemented by VIPR to the case where two viruses, s and t are present.As currently implemented, VIPR only looks for single virus effects. Possible improvements to the software might include the addition of functionality to detect the presence of co-infections and reassortant viruses. This could be accomplished by including among the list of candidates for which likelihoods are computed theoretical combinations of sets of We developed a probabilistic algorithm that relies on a training set of empirical hybridizations that accounts for probe-specific behaviors. Application of this algorithm to a dataset of cultured viruses that cause HF resulted in high accuracy virus identification. Though we report the application of VIPR only in the context of diagnosis of HF, our method of detection is theoretically applicable to any microbial detection problem in which a set of positive and negative control hybridizations is available. Our implementation of a probabilistic algorithm demonstrates the power of a Bayesian approach for discerning important hybridization signals from a complex mixture of nucleic acids. This, in turn, should prove to be of great value as microarray-based diagnostics play more prominent roles in clinical and public health laboratories.VIPR: refers to Viral Identification with a PRobabilistic algorithm; HF: refers to Hemorrhagic fever.AA developed and implemented the algorithm, performed all computational analyses and co-drafted the manuscript. GW and TW optimized and performed all nucleic acid preparations and microarray hybridizations. KF designed the microarray probes. MH and RT prepared virally infected cultures. DW designed the experiments, interpreted the data, conceived of and coordinated the study and co-drafted the manuscript. All authors read and provided feedback for the final version of the manuscript.VIPR output for all 110 HF hybridizations.Click here for file


As part of West Nile (WN) virus surveillance in New York State in 2000, 71,332 ill or dead birds were reported; 17,571 (24.6%) of these were American Crows. Of 3,976 dead birds tested, 1,263 (31.8%) were positive for WN virus. Viral activity was first confirmed in 60 of the state's 62 counties with WN virus-positive dead birds. Pathologic findings compatible with WN virus were seen in 1,576 birds (39.6% of those tested), of which 832 (52.8%) were positive for WN virus. Dead crow reports preceded confirmation of viral activity by several months, and WN virus-positive birds were found >3 months before the onset of human cases. Dead bird surveillance appears to be valuable for early detection of WN virus and for guiding public education and mosquito control efforts.


Alphasatellites are single-stranded molecules that are associated with monopartite begomovirus/betasatellite complexes.Tomato yellow leaf curl China virus (TYLCCNV)/Tomato yellow leaf curl China betasatellite (TYLCCNB) complex. Type 2 was associated with Tobacco curly shoot virus (TbCSV)/Tobacco curly shoot betasatellite (TbCSB) complex. Type 3 was associated with TbCSV/Ageratum yellow vein betasatellite (AYVB) complex. Within each type, nucleotide sequence identity ranged from 83.4 to 99.7%, while 63.4-81.3% identity was found between types. Mixed infections of alphasatellites associated with begomovirus/betasatellite complexes were documented.Alphasatellites were identified in begomovirus-infected plant samples in Yunnan, China. All samples that contained alphasatellites also contained betasatellites, but only some samples that contained betasatellites contained alphasatellites. Thirty-three alphasatellites were sequenced, and they ranged from 1360 to 1376 nucleotides. All alphasatellites contain 3 conserved features: a single open reading frame (Rep), a conserved hairpin structure, and an adenine-rich (A-rich) region. On the basis of the phylogenetic tree of the complete nucleotide sequences, the alphasatellites were divided into 3 types with one exception. Type 1 was associated with Our results validate that alphasatellites are only associated with begomovirus/betasatellite complexes. Thirty-three sequenced alphasatellites isolated from Yunnan Province, China were divided into 3 types--each associated with a specific begomovirus/betasatellite complex. Mix-infections of alphasatellite molecules may not be unusual. Begomovirus in the family Geminiviridae, they are transmitted by the whitefly, Bemisia tabaci [Geminiviruses are a group of plant viruses characterized by their geminate shape and the size of their particles, which encapsidate a circular single-stranded DNA genome. Due to their wide host range and high frequency of genome variation, geminiviruses cause substantial yield losses in many crops, including tomato, cassava, and cotton, throughout tropical and sub-tropical regions worldwide ,2. The ma tabaci . Most bea tabaci ,3. Some a tabaci -9. Analya tabaci .Alphasatellites (formerly DNA1) are circular, single-stranded DNA molecules associated with begomovirus/betasatellite complexes -15. AlphIn China, several begomoviruses are reported to infect squash, tobacco, ageratum, tomato, and malvastrum; many begomovirus isolates are associated with betasatellites, and co-evolution of betasatellites with their helper viruses has been documented ,16-21. IAgeratum conyzoides, Malvastrum coromandelianum, and tobacco, tomato, and squash plants, were collected from widely separated locations in Yunnan. The majority of these isolates were found to be infected with 1 or 2 of the following 7 viruses: Tobacco curly shoot virus (TbCSV) [Tobacco leaf curl Yunnan virus (TbLCYNV) [Tomato yellow leaf curl China virus (TYLCCNV) [Tomato yellow leaf curl Thailand virus (TYLCTHV) [Malvastrum yellow vein virus (MYVV) [Malvastrum yellow vein Yunnan virus (MYVYNV) [Squash leaf curl Yunnan virus (SLCYNV) [Tobacco curly shoot betasatellite (TbCSB) complex (50%) and TYLCCNV/Tomato yellow leaf curl China betasatellite (TYLCCNB) complex (42.9%) were associated with alphasatellites, whereas no samples infected by TYLCTHV/Tomato yellow leaf curl Thailand betasatellite (TYLCTHB), MYVV/Malvastrum yellow vein betasatellite (MYVB), or MYVYNV/Malvastrum yellow vein Yunnan betasatellite (MYVYNB) complexes contained alphasatellites  , Tomato TYLCCNV) , Tomato TYLCTHV) , Malvasts (MYVV) , Malvast(MYVYNV) , and Squ(SLCYNV) . Some ofAJ579345-AJ579361, AJ888445-AJ888455, and FN678899-FN678903  were determined to be 1360 to 1376 nucleotides (nts) in length--this is longer than betasatellites, which range from 1333 to 1355 nts in length. The sequences of these 33 alphasatellites have been submitted to GenBank under the accession numbers Nucleotide sequence comparisons show that the 33 alphasatellites can be divided into 3 types (Table Hibiscus leaf curl virus (HLCA)  from Baoshan District and Y8A-5 from Honghe District. The second subtype consists of 4 alphasatellites  from Honghe District. The third branch consists of 4 alphasatellites, among them, Y8A-6 and Y39A were from Honghe District, and Y240A and Y241A were from Wenshan District. There are 2 subtypes of type 2: one consists of 8 alphasatellites  and the other consists of 2 alphasatellites (Y35A and Y87A-2); all isolates were from Baoshan District. Type 3 molecules consist of 8 alphasatellites  from Baoshan, Honghe, and Yuxi districts, and cluster with All 33 alphasatellites contain 3 conserved features: a conserved hairpin structure, a single open reading frame, and an adenine-rich (A-rich) region Figure . The higA-rich regions are maintained by all alphasatellites immediately downstream of the Rep gene as reported for other alphasatellites. This A-rich region is approximately 153-169 nts long with an A-content of between 52.3-58.4%. The alignment of the sequences of the A-rich region shows that they can be divided into 3 types in accordance with the phylogenetic trees of the complete nucleotide sequences of the alphasatellites /DNA102 (5'-CTGCAGATCCTCCACGTGTATAG-3') or UN101 (5'-AAGCTTGCGACTATTGTATGAAAGAGG-3')/UN102 (5'-AAGCTTCGTCTGTCTTACGAGCTCGCTG-3'), which were designed from the highly conserved regions of the Rep-encoding genes of the determined alphasatellites . BetasatAgeratum yellow vein virus , Cotton leaf curl Multan virus , Hibiscus leaf curl virus , Okra leaf curl virus , and Sida yellow vein Vietnam virus . Nanovirus DNA sequences used for comparisons were Banana bunchy top virus (BBTV AF216221), Faba bean necrotic yellow virus , Milk vetch dwarf virus , and Subterranean clover stunt virus .Sequence data were assembled and analyzed using DNAStar software version 6.0  and MEGA version 4 . SequencThe authors declare that they have no competing interests.YX, PW, PL, HG performed the experiments. YX, PW, XZ involved in data analysis and manuscript preparation. XZ provided overall direction and conducted experimental design, data analysis and wrote manuscript. All authors read and approved the final manuscript.


We identified a novel calicivirus in a pup with enteritis. The isolate was related genetically (90.1% aa identity in the capsid protein) to a lion norovirus strain. Vesivirus genus) and were likely acquired from cats . The virus replicated in experimentally infected dogs and elicited seroconversion, although disease was not reproduced. The virus was antigenically unrelated to feline calicivirus, and antibodies against the virus were identified in 76% of the canine serum specimens collected . By using a broadly reactive primer pair, p289–p290, targeted to highly conserved motives “DYSKWDST” and “YGDD” of the RdRp region of the polymerase complex, we found, unexpectedly, that stool also tested positive for calicivirus  as described by Wang et al. (www.ncbi.nlm.nih.gov/BLAST) and FASTA (www.ebi.ac.uk/fasta33) analysis, we found the highest identity (84.6% nt and 96.7% aa) in a lion norovirus strain, Pistoia/387/06/ITA , is represented in A nucleotide identity plot of the canine norovirus genome 170/07 (from the 3′ end of ORF1 to the poly-A tail) was compared with the lion GIV.2 NoV Pistoia/387/06/ITA and the human GGIV.1 norovirus, Fort Lauderdale/560/98/US (AF414426) . The 3′ Noroviruses are regarded as the major cause of epidemic, nonbacterial gastroenteritis worldwide in humans of all age groups. The viruses are highly contagious and are transmitted by direct contact or by contaminated water and food . RecentlThe pathogenic potential of this novel calicivirus in carnivores remains to be elucidated. In the pup, the norovirus strain was detected in conjunction with a CPV-2a strain that was likely responsible for the severity of the observed clinical signs, since CPV-2 is a major enteric pathogen of dogs. Accordingly, it is difficult to speculate on the pathogenic potential of the novel calicivirus for dogs and experimental infections in gnotobiotic dogs are required. Nevertheless, the norovirus 170/07 was detected in the subject pup for at least 22 days, which indicates active viral replication; the possibility of passive viral transit  in the intestinal tract seems unlikely.In conclusion, our study presents evidence for the existence of a novel calicivirus with enteric tropism that is related genetically to GGIV noroviruses and able to infect dogs. Future large-scale virologic and serologic investigations are needed to assess the ecology of this novel virus in wild and domestic carnivores. Also, because of the extensive social interactions between humans and pets, investigating the zoonotic potential of such animal noroviruses in humans is worthwhile.


A dictionary is a reference book containing an alphabetical list of words, with information about each word; it usually includes meaning, pronunciation, and etymology. The third edition of Brian Mahy’s Dictionary of Virology provides definitions of words commonly and uncommonly used in the field of virology, words that so often divide us into subgroups. Given that new words are associated with new thought processes and the new procedures that accompany them, picking up the latest technical terms and buzzwords is difficult. In addition, these words are added to the astounding number of words we have all accumulated over 10, 20, 30, or 40 years, so that the sum can be overwhelming. Thus, having a book containing all these words is functional and valuable. One can run to a secluded place, check this book, and thereafter appear to know what a graduate student is talking about. Previous editions were available in the United States in hardcover only, but this latest edition is in a convenient paperback form.This book is not a complete dictionary in the classical sense; it does not provide a pronunciation guide as a dictionary might. Mahy prefaces the book with the disclaimer: “Viruses which only infect bacteria, fungi, invertebrates or plants are outside the scope of this Dictionary.” Perhaps the publisher drew the line at 422 pages, or perhaps this book should have been named “Dictionary of (for the most part) Animal Viruses”. In any event, if you are a molecular biologist, clinician, or epidemiologist involved in work with animal viruses, a graduate student, a senior faculty member who is overly specialized, a journalist, or a layman without a life, this book is highly recommended. A Dictionary of Virology includes useful, informative, and concise definitions and brief descriptions of a multitude of words and terms we use each day, and it also contains words used by others. The book’s entries include not only the necessary dry descriptions but also miniexplanations of very complex matters. Key references will lead you in the right direction, should you want to know more.Highlands J virus indicates that this is not the etiologic agent of disease (disease in horses and turkeys illustrates otherwise). The dictionary alleges that lyssa virus is to be a synonym for Rabies virus, but whereas Rabies virus is a lyssavirus (a member of the genus Lyssavirus), there are many lyssaviruses, only one of which is Rabies virus. Trivittatus virus is defined as a member of the “California encephalitis virus serogroup,” but at least this week, it is considered a serotype of the species California encephalitis virus . A few spelling errors occur, which, I am certain, will be corrected in the fourth edition.As might be expected of a book containing this much information, there are some minor shortcomings. For example, names of virus strains are presented for some viruses without explanation as to why names of virus strains are not presented for all viruses. Virus (= species) names are promised as conforming to the latest taxonomic vogue , but not all species names are italicized. The definition for —quite remarkable for a dictionary—pleasant and informative reading. Do not keep this book on your nightstand; by the time you get to it, it will be too late in the day. Instead, carry it in your attaché case or in the glove compartment of your pickup truck. This book reflects the remarkable diversity of viruses and of the terms that have been coined to discuss them so that we may have the requisite common language. I recommend it highly.Never mind the minor and few errors of commission, there are few, if any, (I looked) gross errors of omission. A Dictionary of Virology, therefore, is not only unique, it is useful


The Kato-Katz technique is widely used for the diagnosis of soil-transmitted helminthiasis in epidemiological surveys and is believed to be an inexpensive method. The FLOTAC technique shows a higher sensitivity for the diagnosis of light-intensity soil-transmitted helminth infections but is reported to be more complex and expensive. We assessed the costs related to the collection, processing and microscopic examination of stool samples using the Kato-Katz and FLOTAC techniques in an epidemiological survey carried out in Zanzibar, Tanzania.We measured the time for the collection of a single stool specimen in the field, transfer to a laboratory, preparation and microscopic examination using standard protocols for the Kato-Katz and FLOTAC techniques. Salaries of health workers, life expectancy and asset costs of materials, and infrastructure costs were determined. The average cost for a single or duplicate Kato-Katz thick smears and the FLOTAC dual or double technique were calculated.The average time needed to collect a stool specimen and perform a single or duplicate Kato-Katz thick smears or the FLOTAC dual or double technique was 20 min and 34 sec (20:34 min), 27:21 min, 28:14 min and 36:44 min, respectively. The total costs for a single and duplicate Kato-Katz thick smears were US$ 1.73 and US$ 2.06, respectively, and for the FLOTAC double and dual technique US$ 2.35 and US$ 2.83, respectively. Salaries impacted most on the total costs of either method.The time and cost for soil-transmitted helminth diagnosis using either the Kato-Katz or FLOTAC method in epidemiological surveys are considerable. Our results can help to guide healthcare decision makers and scientists in budget planning and funding for epidemiological surveys, anthelminthic drug efficacy trials and monitoring of control interventions. Ascaris lumbricoides, Trichuris trichiura and the hookworms , might account for a global burden of 39 million disability-adjusted life years (DALYs) lost annually [Chronic infections with one or several of the common soil-transmitted helminths, annually ,2. SchooIn the current era of 'preventive chemotherapy', that is the large-scale administration of anthelminthic drugs to school-aged children and other populations at risk of morbidity , diagnosHere, an economic evaluation of the Kato-Katz and FLOTAC techniques was performed within the frame of an epidemiological survey. Specifically, the costs related to stool collection, transfer to the laboratory, preparation and microscopic examination of single and duplicate Kato-Katz thick smears and the FLOTAC dual or double technique were determined. This information is relevant for researchers and disease control managers for the planning of epidemiological surveys and the monitoring and evaluation of soil-transmitted helminthiasis control programmes.T. trichiura and other soil-transmitted helminths. At the beginning of the study, 1,066 schoolchildren aged between 6 and 20 years, were screened for soil-transmitted helminth infections .This study was carried out on Unguja, the largest island belonging to the Zanzibar archipelago in Tanzania, between March and May 2009. The two primary schools of Kinyasini and Kilombero, located 26 and 32 km north-east of Zanzibar Town, were the selected field sites. Laboratory examinations were conducted in the Helminth Control Laboratory Unguja (HCLU), in Zanzibar Town. The study was readily embedded in a randomised controlled trial, assessing the efficacy and safety of four anthelminthic drug regimens against The study was approved by the ethics committee of Basel, Switzerland  and the Ministry of Health and Social Welfare (MoHSW) of Zanzibar (reference no. ZAMEC/0001/09). Parents or legal guardians of participating children signed a written informed consent sheet. Children consented orally to participate. At the end of the study, all children attending the primary schools of Kinyasini and Kilombero were treated with single oral doses of albendazole (400 mg) and praziquantel (40 mg/kg) free of charge.The headmasters of Kinyasini and Kilombero primary schools were informed about the purpose and procedures of the study. After having obtained written informed consent by the parents/legal guardians and oral consent by the children, the field work was launched. Every morning, starting at 07:00 hours at HCLU, stool containers and collection shelves were loaded onto a 4-wheel drive (4-WD) car, and a team consisting of 4-6 workers from HCLU visited either Kinyasini or Kilombero schools. The team labeled empty containers with unique identification (ID) numbers, distributed these to the children and collected filled containers that had been distributed the day before. Each day, approximately 100 children were enrolled and lime-sized early morning stool samples were collected.A. lumbricoides and T. trichiura and recorded them separately.The filled containers were transferred to HCLU within 2-3 h and processed as follows. Immediately after arrival at HCLU, duplicate Kato-Katz thick smears from each stool sample were prepared by 2-8 members of HCLU, using 41.7 mg templates [Once a Kato-Katz thick smear had been prepared, the stool sample was placed back into the collection shelf ordered by increasing ID. A third of the stool samples was taken and a small amount of stool was weighed to ~1 g using a Kern balance  and preserved in a pre-labeled tube containing 10 ml of 5% formaldehyde. The preserved stool samples were stored at room temperature and examined by the FLOTAC technique within 3 weeks after completion of the clinical trial in late May 2009 .3, specific gravity (s.g.) = 1.20) and 7 .While technicians from HCLU were already acquainted with the Kato-Katz technique, a 3-day training course was offered for the FLOTAC technique, facilitated by two experts from the University of Naples, Italy. During the training, the FLOTAC dual technique was employed, using flotation solutions 4  costs due to materials; and (iii) costs due to infrastructure. We made the following assumptions: screening of 3,000 children for soil-transmitted helminth infections by an experienced research team, with 100 stool samples examined per day in a laboratory (Appendix). The flow of cost determination is shown in Figure To assess the costs for single and duplicate Kato-Katz thick smears and the FLOTAC dual or double technique, the following expenses were determined: (i) costs due to salaries, the salary of each member of the HCLU, according to his or her professional degree, was determined using a questionnaire. Additionally, the time for each working step in the field and in the laboratory was measured several times and averaged. Finally, the number of employees with a specific occupation needed to collect, process and examine 100 stool samples per day using the Kato-Katz or FLOTAC technique was calculated.To assess the For the collection of stool samples in the field the following steps were recorded: (i) the time from departure in the laboratory, school visit and return to the laboratory; (ii) the number of personnel who visited the school; and (iii) the number of stool samples collected per day. Using these data, the average person-time to collect one stool sample was estimated.A. lumbricoides and T. trichiura eggs; (K4) data entry ; (K5) data entry ; and (K6) washing and preparing materials for the next day.The duration of each distinct step in the laboratory needed to perform the Kato-Katz or FLOTAC technique was measured and averaged Table . For dupA. lumbricoides, hookworm and T. trichiura eggs; (F8) reading chamber 2 of FLOTAC apparatus quantitatively for A. lumbricoides, hookworm and T. trichiura eggs; (F9) data entry ; (F10) data entry ; and (F11) washing and preparing materials for the next day.For the FLOTAC technique the following steps were included: (F1) weighing ~1 g of stool and homogenization in 10 ml of 5% formaldehyde; (F2) filtering homogenized stool and transfer into two 15-ml Falcon tubes labeled with personal ID; (F3) centrifugation of Falcon tubes, discarding supernatant and filling FS4 into tubes; (F4) assembly of FLOTAC apparatus; (F5) filling each chamber of the FLOTAC apparatus with the homogenized stool suspension (pellet and FS4) from one of the two Falcon tubes; (F6) centrifugation and translation of FLOTAC apparatus; (F7) reading chamber 1 of FLOTAC apparatus quantitatively for The time to prepare and read a single Kato-Katz thick smear was assessed as follows: K1a) the time needed to label duplicate microscope slides was divided by 2  materials that can only be used once ; (ii) materials that can be reused (e.g. microscope slides and pipettes); and (iii) materials that have a long life expectancy (e.g. microscope and centrifuge). The frequency of possible re-use was determined in the field and laboratory. Additionally, the effort to render the materials re-usable was accounted for as a working step (e.g. washing). The life duration of materials with a longer life expectancy was estimated by consulting laboratory experts and the literature. For these materials, it was estimated how many days per year they were to be used. All materials, asset costs, life time, days in use and total costs are listed in Table To estimate the total Costs due to infrastructure originated from (i) the use of the laboratory building for stool examinations ; and (ii) the use of the car for stool container distribution and collection in schools. The monthly costs for the laboratory and the asset costs for the 4-WD car, together with the monthly expenditures for car maintenance were reported by the head of HCLU. The life expectancy of a car was derived by consulting WHO-choice [O-choice  and the costs due to materials; (iv) petrol costs; (v) costs due to infrastructure; and (vi) costs without collection of stool samples in the field.A series of one-way sensitivity analyses were performed to determine the robustness of the cost estimations and to assess to what extent the costs vary if a specific parameter changes -24. We aT. trichiura, hookworm and A. lumbricoides was 62.8%, 19.8% and 9.2%, respectively. The FLOTAC double technique was performed on 343 stool samples and revealed respective prevalences of 67.1%, 11.7% and 10.2%. Infection intensities, according to WHO guidelines [T. trichiura, hookworm and A. lumbricoides eggs was 88%, 81% and 68%, respectively. The respective sensitivities for FLOTAC were 95%, 54% and 88%.Among 1,066 school children screened with duplicate Kato-Katz thick smears, the prevalence of idelines , were moIn 2009 the daily salary of an employee at HCLU was, on average, US$ 18.83 .The average person-time needed to collect one stool sample in the field, including transfer to HCLU was 10 min and 55 sec (10:55 min) , the costs of the laboratory building were US$ 0.13 per stool sample. The 4-WD car was bought in 2002 for US$ 22,000. WHO-choice suggests a lifetime for a 4-WD car of 8 years . Considecosts due to salaries, costs due to materials and costs due to infrastructure, the total expenses were US$ 1.73 for a single Kato-Katz thick smear, US$ 2.06 for duplicate Kato-Katz thick smears, US$ 2.35 for the FLOTAC double technique and US$ 2.83 for the FLOTAC dual technique  to 25.9% (single Kato-Katz thick smear). Doubling the petrol costs raised the total costs by 5.3%  to 8.6% (single Kato-Katz thick smear).The one-way sensitivity analyses illustrate the effect of alternated cost variables on the total costs of either diagnostic approach Table . If salaIn a scenario where stool samples are not collected in the field .The time requirements and costs for the diagnosis of soil-transmitted helminth infections in a cross-sectional epidemiological survey, using either the Kato-Katz technique performed on fresh stool samples or the FLOTAC technique using preserved stool samples, are considerable. Indeed, in our study, the costs for a single or duplicate Kato-Katz thick smears were US$ 1.73 and US$ 2.06, respectively. As expected, the costs for the FLOTAC technique were even higher: US$ 2.35 for the FLOTAC double technique and US$ 2.83 for the FLOTAC dual technique. The higher costs of the FLOTAC technique are mainly caused by the longer preparation time for stool samples and microscopic examinations in the laboratory. Previous studies have shown that the FLOTAC technique is more sensitive than the Kato-Katz method for the diagnosis of soil-transmitted helminth infections -18, and In view of the high costs to collect a single stool sample in the field and subsequent examination in the laboratory, preventive chemotherapy without prior diagnosis, as advocated by WHO for high-risk groups in endemic settings is, at first sight, justified ,26. In eA. lumbricoides and T. trichiura. While this procedure increases costs, it results in higher sensitivity of the Kato-Katz method and might explain the differences in the diagnostic performance of the Kato-Katz and FLOTAC techniques compared to previous comparative investigations [It is important to note that in our study, the Kato-Katz method was performed by rigorously adhering to the WHO bench aids . Indeed,igations -18. RoutMoreover, our data are derived from a large survey, where many stool samples were collected and processed simultaneously. One-way sensitivity analyses showed that costs decrease by more than 50% if, for example, individual patients are managed at hospital or public health centre laboratories, without a need for a field team and a 4-WD car. However, in those laboratories, stool samples are not necessarily examined in large numbers, and hence cost and time are likely higher than presented here, due to economy of scale issues . Of noteA. lumbricoides and hookworm were low and infection intensities of all soil-transmitted helminth species were light. Hence, the counting of helminth eggs by microscopists was relatively quick. In settings with higher prevalence and infection intensities, quantitative microscopic examination will be more time consuming and costs will rise.Furthermore, in our setting, the prevalences of Of additional cost relevance is that salaries usually constitute the largest part of the total service costs . Our senFasciola hepatica in rats [Trichuris infections revealed a preparation and reading time of only 09:48 min [In our study, the time to prepare and microscopically examine a stool sample using the FLOTAC double technique was 17:19 min. This result is in line with the time of 21 min reported in a recent study, where the FLOTAC technique was applied for the diagnosis of 9:48 min . Of noteAnother potential bias in our study is that that the laboratory workers were constantly supervised by an external researcher and that their working speed was recorded. It is hence possible that the staff's working speed differed from normal activity . It is aDespite that the cost estimates for the Kato-Katz and FLOTAC method from our study are not readily transferable to other epidemiological settings where additional costs might occur, or costs might be saved, we believe that they give a reasonable idea of the expenses related to soil-transmitted helminth diagnosis and can guide scientists and decision makers in budget planning for epidemiological surveys. The real costs of diagnosis are of considerable relevance to large-scale control programmes, which often need to balance the costs of treatments against the costs of diagnosis. As global funds to control soil-transmitted helminth infections become more widely available this will be an increasingly important issue to ensure that control programmes are cost-effective and sustainable.G. Cringoli is the inventor and current patent holder of the FLOTAC apparatus. In case the currently ongoing validation of the FLOTAC technique for human and veterinary diagnosis will be successful, the method will be licensed free of charge to the WHO and interested public non-commercial research centers. None of the other authors has any conflict of interest concerning the work reported in this paper.BS, SK, KAM, GC and JU designed the study; BS, SK, KAM, ISK, LR, DR and JU implemented the study; BS managed the data; BS, SK and JU analysed and interpreted the data; BS wrote the first draft of the paper; SK, KAM, ISK, LR, GC, DR and JU revised the manuscript. All authors read, and approved the manuscript prior to submission and assisted with the final revision of the manuscript.