We describe an external quality assurance (EQA) study designed to assess the efficiency and accurateness of molecular and serological methods used by expert laboratories performing YF diagnosis.
For molecular diagnosis evaluation, a panel was prepared of 14 human plasma samples containing specific RNA of different YFV strains (YFV-17D, YFV South American strain [Brazil], YFV IvoryC1999 strain), and specificity samples containing other flaviviruses and negative controls. For the serological panel, 13 human plasma samples with anti-YFV-specific antibodies against different strains of YFV (YFV-17D strain, YFV IvoryC1999 strain, and YFV Brazilian strain), as well as specificity and negative controls, were included.
Thirty-six laboratories from Europe, the Americas, Middle East, and Africa participated in these EQA activities. Only 16% of the analyses reported met all evaluation criteria with optimal performance. Serial dilutions of YFV-17D showed that in general the methodologies reported provided a suitable sensitivity. Failures were mainly due to the inability to detect wild-type strains or the presence of false positives. Performance in the serological diagnosis varied, mainly depending on the methodology used. Anti-YFV IgM detection was not performed in 16% of the reports using IIF or ELISA techniques, although it is preferable for the diagnosis of YFV acute infections. A good sensitivity profile was achieved in general; however, in the detection of IgM antibodies a lack of sensitivity of anti-YFV antibodies against the vaccine strain 17D was observed, and of the anti-YFV IgG antibodies against a West African strain. Neutralization assays showed a very good performance; however, the unexpected presence of false positives underlined the need of improving the running protocols.
This EQA provides information on each laboratory's efficacy of RT-PCR and serological YFV diagnosis techniques. The results indicate the need for improving serological and molecular diagnosis techniques and provide a follow-up of the diagnostic profiles.
Yellow fever (YF) is a viral disease which is symptomatic in about 5–20% of infected people. In its mildest form YF is characterized by non-specific fever and headache, while the severe form of the disease is characterized by high fever, jaundice, bleeding, and eventually shock and multiple organ failure. Among those who develop severe disease 20–50% may die. To date, there are no antivirals for specific treatment of the infection, and YF vaccination of the population at risk is the best approach to prevent and control the disease [1] , [2] .
Yellow fever virus (YFV) is transmitted to humans by bites of infected mosquitoes. In the jungle setting, mosquitoes and monkeys maintain the virus via an enzootic cycle. The virus can also be transmitted vertically from female mosquitoes to eggs as a maintenance mechanism from one year to the next. A second transmission cycle, the urban cycle, involves humans and the mosquito species Aedes (Stegomyia) aegypti. An intermediary cycle is maintained in humid and semi-humid African savannas with viral transmission to humans and to non-human primates through different Aedes sp. Mosquito control is not possible in areas of sylvatic transmission, thus eradication of YF is not a plausible option [3] .
YF remains an important public health problem for people in endemic regions in Africa and South America and is considered an emerging disease, with a dramatic upsurge in the number of cases in recent years [4] . YF cases occur every year in Africa. For example, during 2011 cases from Senegal, Sierra Leone, Uganda, and Ivory Coast have been reported to the World Health Organization (WHO) (
The clinical diagnosis of YF and the identification of YFV vaccine-associated adverse events (YFVAE) are particularly difficult because of similar symptoms of a wide range of diseases (e.g. dengue, leptospirosis, viral hepatitis, malaria, hemorrhagic viral diseases), therefore laboratory confirmation is essential. As criteria for laboratory YF diagnosis, WHO recommends the detection of YFV-specific IgM or a fourfold or greater rise in serum IgG levels in the absence of recent YF vaccination and a negative diagnosis for other flaviviruses. Isolation of YFV, positive post-mortem liver histopathology, detection of YFV antigen in tissues by immunohistochemistry, or detection of YFV genome in blood or organs by RT-PCR also confirm the presence of YFV infection [7] . Recently, we have reported the detection of YFV-17D genome in the urine of healthy YF vaccinees and vaccinees with serious post- vaccination adverse events, which seems very promising for the investigation in outbreak situations by non-invasive sampling methods [8] .
Molecular methods for the detection of the viral genome offer a rapid, sensitive, and highly specific alternative for early serological diagnosis during the viraemic phase of infection or in post-mortem tissues. After the viraemic phase the use of serological methods represents a good option to confirm the infection, but generally two samples are required to be taken at least two weeks apart. Serological diagnosis commonly includes the use of haemagglutination, ELISA, indirect immunofluorescence (IIF) and seroneutralization assays, but flavivirus serological cross-reactions constitute a major obstacle in achieving confirmed diagnoses or reliable serosurveys in endemic areas where other flaviviruses circulate (e.g. dengue, Zika, St. Louis encephalitis, or West Nile viruses). Seroneutralization is considered as the most specific serological technique. However, the assays are laborious and time consuming and are only available in expert laboratories. But the availability of commercial assays for the serological diagnosis of YF has increased the implementation of such techniques. Commercial assays are, in general, thoroughly standardized and offer good standards of sensitivity, specificity, and reproducibility in diagnostic laboratories in order to receive the authorisation for in vitro diagnostics.
The performance of the different techniques applied in YF diagnosis may vary between laboratories, and so far no external quality assessment (EQA) studies addressing their accomplishment have been performed. This international EQA is an important tool to evaluate the performance of protocols currently in use in diagnostic laboratories and to highlight weaknesses in their methodologies and operating procedures.
Institutions involved in laboratory diagnostics of YFV infection were invited to participate in this study. Invitees consisted of members of the European Network of “Imported” Viral Diseases (ENIVD), national/regional YFV reference laboratories, and diagnostic laboratories contacted through the Pan American Health Organization (PAHO) or the African Network of Laboratories for polioviruses and hemorrhagic fevers diagnosis. The study was announced as an EQA study on YFV molecular and serological diagnostic methods proficiency, which included certifying and publishing the results in a comparative and anonymous manner.
Thirty-six laboratories from Europe (n = 28), the Americas (n = 7), Middle East Asia (n = 1), and Africa (n = 1) participated in these EQA activities, and reports including 32 and 31 data sets of results were returned for the molecular (28 laboratories) and serological (28 laboratories) diagnosis EQAs, respectively.
Twenty laboratories participated both in the molecular diagnostics EQA and the serological diagnostics EQA from Europe (n = 15), the Americas (n = 4), and Africa (n = 1): IRBA-IMTSSA, Marseille, France; Bernhard-Nocht-Institut, Hamburg, Germany; Institut für Mikrobiologie der Bundeswehr, Munich, Germany; Aristotle University of Thessaloniki, School of Medicine, Greece; Istituto Nazionale per le Malattie Infettive “L. Spallanzani”, Rome, Italy; Fondazione IRCCS Policlinico San Mateo, Pavia, Italy; Norwegian Institute of Public Health, Oslo, Norway; CEVD/INS, Aguas de Moura, Portugal; Institute of Microbiology and Immunology, University of Ljubljana, Ljubljana, Slovenia; Instituto de Salud Carlos III, Madrid, Spain; Hospital Clínic i Provincial de Barcelona, Barcelona, Spain; Spiez Laboratory, Spiez, Switzerland; University of Geneva, Geneva, Switzerland; Erasmus Medical Centre, Rotterdam, The Netherlands; Health Protection Agency, CEPR, Porton Down, Salisbury, United Kingdom; Centro Nacional de Enfermedades Tropicales, CENETROP, Santa Cruz, Bolivia; Instituto Nacional de Salud, Bogotá, Colombia; Institut Pasteur de la Guyane, Cayenne Cedex, French Guiana; Laboratorio Central de Salud Pública, Asunción, Paraguay; Special Pathogens Unit, National Institute for Communicable Diseases, National Health Laboratory Service, SPU/NICD-NHLS, Johannesburg, South Africa.
Eight laboratories participated exclusively in the YFV molecular diagnostics EQA, from Europe (n = 7) and the Middle East (n = 1): Institute of Virology, Medical University Vienna, Vienna, Austria; Slovak Academy of Science, Bratislava, Slovakia; Assistance Publique-Hôpitaux de Marseilles, Hôpital de la Timone, AP-HM TIMONE, Marseille, France; Army Medical and Veterinary Research Center, Rome, Italy; Centre for Biothreat Preparedness, Helsinki, Finland; Universitätsklinikum Freiburg, Freiburg, Germany; Institut für Virologie, Marburg, Germany; National Center for Zoonotic Viruses, MOH-PHL, Tel-Hashomer, Israel.
Eight laboratories participated exclusively in the serological diagnosis EQA, from Europe (n = 5) and the Americas (n = 3); Instituut voor Tropische Geneeskunde, Antwerp, Belgium; Institute of Public Health, Ostrava, Czech Republic; Euroimmun AG, Lübeck, Germany; National Center for Epidemiology, Budapest, Hungary; Crucell Switzerland AG, Berne, Switzerland; Bio Manguinhos, Rio de Janeiro, Brazil; Viral Zoonoses National Microbiology Laboratory, Winnipeg, Manitoba, Canada; Instituto Nacional de Higiene y Medicina Tropical “LIP”, Guayaquil, Ecuador.
The European Network for the Diagnostics of ‘Imported’ Viral Diseases -Collaborative Laboratory Response Network (ENIVD-CLRN) established and coordinated this EQA as in other EQAs previously performed [9] – [12] .
The molecular diagnosis EQA panel consisted of inactivated YFV preparations generated from Vero E6 cell culture supernatants infected with different YFV strains: the vaccine strain (YFV-17D), a South American strain (Brazil), and a West African strain (IvoryC1999) [13] . Supernatants were inactivated by heating for 1 h at 56°C and by gamma irradiation (25 kilogray [kGy]) to ensure their non-infectivity. The inactivated supernatant viral load was estimated after heat inactivation and additionally after gamma irradiation by an in-house real-time RT-qPCR with a 95% detection limit in copy number estimated in 6.48 copies/reaction (rxn) (95% CI: 2.35–235 copies/rxn) (C. Domingo, unpublished results).
The inactivated material was diluted in serum plasma to prepare a set of ten positive samples that included five serial 10-fold dilution series of YFV-17D (3×10 6 Genome equivalents [GE]/sample to 3×10 2 GE/sample), two YFV (Brazil) dilutions (10 4 GE/sample and 10 3 GE/sample), and three YFV (strain IvoryC1999; GenBank Acc. No.: AY603338) dilutions (2×10 4 GE/sample to 69 GE/sample). As specificity controls we prepared two additional plasma samples, one of them containing West Nile virus (WNV [New York]), Japanese encephalitis virus (JEV [strain SA-14-02]), St. Louis encephalitis virus (SLEV [Parton]), and tick-borne encephalitis virus (TBEV [strain Absettarov]). The second plasma sample contained the four dengue serotypes (DENV-1 VR344 [strain Thai 1958], DENV-2 VR345 [TH-36 strain], DENV-3 VR216 [H87 strain], and DENV-4 VR217 [H241 strain]). Two negative control plasma samples were also included ( Table 1 ).
| Sample ID | #2 | #9 | #12 | #4 | #14 | #10 | #5 | #13 | #1 | #6 | #11 | #3 | #8 | #7 | ||||
| Viral load in sample | 3×10 6 | 3×10 5 | 3×10 4 | 3×10 3 | 3×10 2 | 10 4 | 10 3 | 2×10 4 | 2×10 3 | 69 | NEG | NEG | NEG | NEG | ||||
| Lab. no. | RT-PCR technique | 17D | 17D | 17D | 17D | 17D | Brazil | Brazil | Ivory Coast | Ivory Coast | Ivory Coast | SLEV/JEVWNV/TBEV | DENV1-4 | neg | neg | TARGET | Score * | Classification |
| 16a | TaqMan RT-PCR a | + | + | + | + | + | + | + | + | + | (−) | − | − | − | − | YFV | 26 | OPTIMAL |
| 8 | TaqMan RT-PCR b | + | + | + | + | (−) | + | + | + | + | (−) | − | − | − | − | PANFLAV | 24 ¶ | OPTIMAL |
| 17b | RT nested PCR c | + | + | + | + | + | + | + | + | + | + | − | (+) | − | − | PANFLAV | 24 ¶ | NON OPTIMAL |
| 1 | TaqMan RT-PCR a | + | + | + | + | + | + | + | + | + | + | − | (+) | − | − | YFV | 24 | NON OPTIMAL |
| 27 | TaqMan RT-PCR d | + | + | + | + | + | + | + | + | + | + | − | (+) | − | − | YFV | 24 | NON OPTIMAL |
| 28 | TaqMan RT-PCR e | + | + | + | + | + | + | + | + | + | + | − | (+) | − | − | YFV | 24 | NON OPTIMAL |
| 15 | TaqMan RT-PCR a | + | + | + | + | + | + | + | + | + | (−) | − | (+) | − | − | YFV | 22 | NON OPTIMAL |
| 17a | TaqMan RT-PCR f | + | + | + | + | + | + | + | + | + | (−) | − | (+) | − | − | YFV | 22 | NON OPTIMAL |
| 6 | RT nested PCR x | + | + | (−) | (−) | (−) | + | + | + | + | (−) | − | − | − | − | PANFLAV | 20 ¶ | OPTIMAL |
| 22a | RT nested PCR g | + | + | ND | + | (−) | + | + | (−) | (−) | + | − | − | − | − | PANFLAV | 20 ¶ | OPTIMAL |
| 16b | Heminest Rt-PCR c | + | + | + | + | + | + | + | + | + | (−) | − | (+) | − | − | PANFLAV | 20 ¶ | NON OPTIMAL |
| 2 | TaqMan RT-PCR a | + | + | + | + | (−) | + | + | + | + | (−) | − | (+) | − | − | YFV | 20 | NON OPTIMAL |
| 9 | TaqMan RT-PCR a | + | + | + | + | (−) | + | + | + | + | (−) | − | (+) | − | − | YFV | 20 | NON OPTIMAL |
| 14 | RT nested PCR g | + | + | + | (−) | (−) | + | (−) | + | (−) | (−) | − | − | − | − | PANFLAV | 18 ¶ | OPTIMAL |
| 4 | TaqMan RT-PCR a | + | + | + | + | (−) | + | + | + | (−) | (−) | − | (+) | − | − | YFV | 18 | NON OPTIMAL |
| 10 | TaqMan RT-PCR e | + | + | + | + | + | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 14 | NON OPTIMAL |
| 11 | RT-PCR h | + | + | + | + | + | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 14 | NON OPTIMAL |
| 20 | Heminest RT-PCR c | + | + | + | + | + | + | + | + | + | + | (+) | (+) | − | (+) | PANFLAV | 16 ¶ | NON OPTIMAL |
| 3b | TaqMan RT-PCR e | + | + | + | + | + | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 14 | NON OPTIMAL |
| 5 | TaqMan RT-PCR x | + | + | + | + | + | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 14 | NON OPTIMAL |
| 13 | TaqMan RT-PCR a | + | + | ND | + | + | (−) | (−) | + | (−) | (−) | − | (+) | − | − | YFV | 14 | NON OPTIMAL |
| 18 | TaqMan RT-PCR e | + | + | + | + | + | (−) | (−) | (−) | (−) | (−) | (+) | − | − | − | YFV | 14 | NON OPTIMAL |
| 3a | RT nested PCR f | + | + | + | (−) | (−) | + | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 12 | NON OPTIMAL |
| 19 | TaqMan RT-PCR a | + | + | + | (−) | (−) | + | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 12 | NON OPTIMAL |
| 22b | TaqMan RT-PCR e | + | + | ND | + | + | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 12 | NON OPTIMAL |
| 7 | TaqMan RT-PCR i | + | + | + | + | + | + | (−) | (−) | (−) | + | (+) | (+) | − | (+) | YFV | 10 | NON OPTIMAL |
| 21 | TaqMan RT-PCR e | + | + | + | (−) | (−) | (−) | (−) | (−) | (−) | (−) | − | (+) | − | − | YFV | 10 | NON OPTIMAL |
| 25 | RT-nested PCR x | + | (−) | (−) | (−) | (−) | ND | (−) | (−) | ND | (−) | − | − | − | − | YFV | 8 | NON OPTIMAL |
| 12 | SYBR-RT-PCR b | + | + | + | (−) | (−) | + | (−) | (−) | + | (−) | (+) | (+) | (+) | − | PANFLAV | 6 | NON OPTIMAL |
| 23 | RT-PCR j | + | + | + | (−) | (−) | + | (−) | (−) | (−) | (−) | − | (+) | − | − | PANFLAV | 0 | NON OPTIMAL |
| 24 | RT-nested PCR k | (−) | (−) | (−) | (−) | (−) | (−) | (−) | (−) | (−) | (−) | − | − | − | − | YFV | NS | NON OPTIMAL |
| 26 | RT-nested PCR f | + | (−) | (−) | + | (−) | (−) | + | (−) | + | + | (+) | (+) | (+) | (+) | YFV | NS | NON OPTIMAL |
| CORRECT RESULTS (%) | 96.8 | 90.6 | 86.2 | 71.8 | 53.1 | 67.7 | 50 | 50 | 48.3 | 25 | 84.3 | 25 | 93.7 | 93.7 |
The score reflects the quality of the diagnostic results: +, positive result (score 2 points); (+), false positive (score -2 points); −, correct negative result (score 2 points); (−), false negative (score 0 points).
[20] ;
adapted from [23] ;
[19] ;
[24] ;
[21] ;
in-house;
[18] ;
[22] ;
[16] ;
[15] ;
[17] ;
not provided.
Sequencing performed; NS: Negative Score; ND: not done; PANFLAV: panflavivirus; YFV: yellow fever virus.
Sample preparations were tested by an in-house real-time quantitative RT-PCR to validate the quality of the samples.
For the serological diagnosis, a panel of 13 samples was prepared by diluting anti-YFV-positive sera from YF vaccinees and from wild-type YFV infections with fresh frozen plasma previously confirmed as negative for flaviviruses. After dilution, the samples were heat inactivated (56°C, 1 h). The proficiency panel consisted of a set of nine samples which included four serial 2-fold dilutions of YFV-17D-positive sera (IgM and IgG positive), two dilutions from a West African wild-type YFV infection serum (IgM negative, IgG positive), and three dilutions from a positive South American wild-type YFV infection serum (IgM and IgG positive). As specificity controls, we included aliquots of two sera containing IgM and IgG antibodies reactive for other flaviviruses (WNV, DENV) and two additional negative sera as controls ( Table 2 ).
| Sample ID | #6 | #11 | #7 | 4# | #5 | #10 | #14 | #13 | #1 | #2 | #9z | #8 | #12 | |||
| αYF17D | αYF17D | αYF17D | αYF17D | αYF-AFR | αYF-AFR | αYF-SA | αYF-SA | αYF-SA | αWNV | αDEN | neg | neg | ||||
| Dilution | 1∶5 | 1∶10 | 1∶20 | 1∶40 | 1∶10 | 1∶20 | 1∶20 | 1∶40 | 1∶80 | 1∶20 | 1∶20 | Score* | ||||
| Lab. no. | Assay | IgM/IgG+/+ | IgM/IgG+/+ | IgM/IgG+/+ | IgM/IgG+/− | IgM/IgG−/+ | IgM/IgG−/− | IgM/IgG+/+ | IgM/IgG+/+ | IgM/IgG+/+ | IgM/IgG−/− | IgM/IgG−/− | IgM/IgG−/− | IgM/IgG−/− | IgG | IgM |
| 18 | IIF a | −/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | +/+ | −/+ | −/− | −/− | −/− | −/− | 22 | 16 |
| 12b | IIF a | −/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | +/+ | +/+ | −/− | −/− | −/− | −/− | 22 | 18 |
| 27 | IIF | +/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | +/+ | +/+ | −/− | −/− | −/− | −/− | 22 | 20 |
| 4 | IIF a | −/+ | −/+ | −/+ | −/− | −/− | −/− | +/+ | +/+ | +/+ | −/− | −/+ | −/− | −/− | 20 | 18 |
| 6 | IIF a | −/+ | −/− | −/eq | −/− | −/− | −/− | +/+ | +/+ | eq/+ | −/eq | −/− | −/− | −/− | 20 | 18 |
| 7 | IIF c | −/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | +/+ | eq/+ | −/− | −/− | −/− | −/+ | 20 | 18 |
| 8 | IIF a | +/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | −/+ | +/− | −/− | −/− | −/− | −/− | 20 | 18 |
| 29 | IIF a | +/+ | −/+ | −/− | −/− | −/− | −/− | +/+ | +/+ | +/− | −/− | −/− | −/− | −/− | 20 | 20 |
| 10 | IIF b | +/+ | +/+ | +/+ | −/+ | −/− | −/− | +/+ | +/− | −/− | −/− | −/− | −/− | −/− | 20 | 22 |
| 5 | IIF b | +/+ | +/− | −/− | −/− | −/− | −/− | +/+ | +/+ | +/+ | −/− | −/− | +/− | +/+ | 18 | 18 |
| 20a | IIF | +/+ | −/− | −/− | −/− | −/− | −/− | +/+ | +/+ | −/− | −/− | −/− | −/− | −/− | 18 | 18 |
| 3 | IIF a | +/− | +/− | +/− | −/− | −/− | −/− | +/+ | +/+ | +/+ | −/− | −/− | +/− | −/− | 18 | 22 |
| 2 | IIF b | nd/+ | nd/+ | nd/− | nd/− | nd/− | nd/− | nd/+ | nd/+ | nd/+ | nd/+ | nd/+ | nd/− | nd/− | 18 | nd |
| 25 | IIF a | +/+ | eq/− | −/− | −/eq | −/− | −/− | +/+ | +/+ | eq/− | eq/− | −/− | eq/− | −/− | 16 | 18 |
| 12a | IIF b | nd/− | nd/− | nd/− | nd/− | nd/− | nd/− | nd/+ | nd/+ | nd/− | nd/− | nd/− | nd/− | nd/− | 16 | nd |
| 13 | IIF d | nd/− | nd/− | nd/− | nd/− | nd/− | nd/− | nd/+ | nd/+ | nd/− | nd/− | nd/− | nd/− | nd/− | 16 | nd |
| 28 | IIF a | −/− | −/− | −/− | −/− | −/− | −/− | +/+ | +/− | −/− | −/− | −/− | −/− | −/− | 14 | 16 |
| 14a | IIF a | +/− | −/− | −/− | −/− | −/− | −/− | +/+ | +/− | +/− | −/− | −/− | −/− | −/− | 14 | 20 |
| 11 | IIF b | nd/− | nd/− | nd/− | nd/− | nd/− | nd/− | nd/+ | nd/− | nd/− | nd/− | nd/− | nd/− | nd/− | 14 | nd |
| 1 | IIF a | −/− | −/− | −/− | −/− | −/− | −/− | +/− | +/− | −/− | −/− | −/− | −/− | −/− | 12 | 16 |
| 16 | ELISA f , g | −/− | −/− | −/− | −/− | −/− | −/− | +/− | +/− | +/− | −/− | −/− | −/− | −/− | 12 | 18 |
| 19 | ELISA b | +/eq | +/− | +/+ | eq/+ | −/− | −/− | +/− | +/− | −/+ | +/+ | −/+ | +/+ | −/+ | 10 | 20 |
| 26 | ELISA b | −/nd | −/nd | −/nd | nd/nd | −/nd | −/nd | nd/nd | +/nd | −/nd | −/nd | −/nd | −/nd | −/nd | nd | 14 |
| 23 | ELISA | −/nd | −/nd | −/nd | −/nd | −/nd | −/nd | +/nd | +/nd | +/nd | −/nd | −/nd | −/nd | −/nd | nd | 18 |
| 22 | ELISA e | eq/nd | eq/nd | −/nd | −/nd | −/nd | −/nd | +/nd | +/nd | +/nd | −/nd | −/nd | −/nd | −/nd | nd | 22 |
| 24 | NT | + | + | + | + | − | − | + | + | + | − | − | − | − | 24 | |
| 30 | NT | + | + | + | + | + | − | + | + | + | − | − | − | + | 24 | |
| 9 | NT | + | + | + | − | − | − | + | − | − | − | − | − | − | 18 | |
| 15 | NT | + | + | + | − | + | − | − | + | + | − | − | + | + | 18 | |
| 14b | NT | + | + | − | − | − | − | + | − | − | − | − | − | − | 16 | |
| 20b | HAI | <1∶20 | <1∶20 | <1∶20 | <1∶20 | <1∶20 | <1∶20 | 1∶20 | <1∶20 | <1∶20 | <1∶20 | <1∶20 | <1∶20 | <1∶20 | 6 |
The score reflects the quality of the diagnostic results: +, positive results; −, negative result; nd, not done; eq, equivocal. NT, seroneutralization test; HAI, haemagglutination inhibition test; Wrong results are marked in grey.
EUROIMMUN;
In-house assay;
[25] ;
Bernhard Nocht Institut für Tropenmedizin, Hamburg;
[32] ;
[33] ;
[34] .
For both panels, aliquots of 100 µl each were number-coded, freeze dried for 24 h (Christ, AlphaI-5, Hanau, Germany) and stored at 4°C until dispatch.
To validate the molecular panel, we tested three different sets of EQA samples before distribution by the Robert Koch Institute (RKI), Berlin, Germany. After reconstitution with 100 µl of water, the samples were extracted using the QIAamp viral RNA minikit (Qiagen, Hilden, Germany) according to the manufacturer's instructions. As mentioned above, we estimated the YFV genome copies present in these samples by an in-house real-time RT-PCR ( Table 1 ).
Similarly, two sets of samples for the serological EQA panel were validated before distribution. After reconstitution with 100 µl of water, samples were analyzed by IIF for the presence of specific YFV antibodies using commercially available kits (FK 2665-1010-G and FK 2665-1010-M, Anti-yellow fever virus IIFT, Euroimmun, Lübeck, Germany) following the manufacturer's instructions. These assays have demonstrated a specificity of 94.7% and 96.7% in the detection of IgG and IgM antibodies in two panels of patient sera involving 300 and 294 sera each. Similarly, their sensitivity was determined as 94.7% for IgG and 94.4% for IgM (stated by manufacturer). A microneutralization assay in Vero E6 cells was also carried out to confirm the presence and specificity of the antibodies as previously described [14] .
The EQA panels were distributed to participants with full instructions. Samples were shipped at ambient temperature by post to participating laboratories. We requested participant laboratories to resuspend the samples in 100 µl of water and to analyse the material as serum samples for YFV molecular/serological diagnosis as done routinely. They were asked to report their results and any problems encountered, as well as to provide information on the protocol details (RT-PCR method, extraction procedure, serological methods, sera dilution) using a common form included in the documentation.
To assure anonymous participation, an individual numerical identification code was assigned to the results sent by each laboratory. This number was followed by a letter (a, b) in case different laboratory results were received based on different methods.
A scoring system was established for sensitivity and specificity obtained by each participant laboratory. For the molecular diagnostics EQA evaluation we assigned two points for correct results (100% = 28), and penalised false-positive results with -2 points. We considered those methods as non-optimal which failed to detect one or more strains of YFV, or presented false-positive results in the negative samples. In those cases when a false-positive amplification result was obtained by RT-PCR in the “non-specificity control” samples (#3 and #11), which were however correctly identified by sequencing, the result was considered correct.
For the serological diagnostics EQA evaluation we also assigned two points for correct results (100% = 26), whereas false-negative/-positive results were not scored. Equivocal or borderline results were considered as positive. IgM and IgG results were considered separately.
The complete panel of results was sent to the participants in an anonymous manner where they could only identify the results from their own laboratory.
Data collected were entered into Microsoft Excel (Microsoft Corp., Bellingham, WA, USA) and analysed using SPSS 14.0 for Windows (SPSS Inc., Chicago, IL, USA).
Logit analysis was used to evaluate the effect of viral RNA concentration on the RT-PCR performance by using cumulative fractions of positive results reported for each test sample of the 10-fold dilution series of YFV-17D. The result reflects the performance of a hypothetical average laboratory.
Results with respect to categorised variables were analysed by McNemar's test. T-test and Mann-Whitney tests were used to estimate the effect of the real-time RT-PCR format on the performance. P-value<0.05 was considered to indicate statistical significance.
A total of 36 laboratories participated in this EQA. Among the participants, 20 laboratories (56%) reported both serological and molecular results, indicating that they included both approaches in their diagnostic algorithm. However, 22% (8 out of 36) of the participants only applied either molecular or serological techniques, respectively.
A total of 32 laboratory results were received for the molecular diagnosis study ( Table 1 ), including four double sets from laboratories using two methods each (sets 3ab, 16ab, 17ab, 22ab). A total of 31 laboratory results were received for the serological diagnosis study ( Table 2 ), including three double sets from laboratories using two methods each (sets 12ab, 14ab, 20ab).
A variety of tests were used for screening and identification of YFV genome by participating laboratories; these included RT-PCR (n = 2, 6% of the laboratory results) [15] , [16] , RT-nested PCR (n = 8, 25%) [15] , [17] , [18] , hemi-nested RT-PCR (n = 2, 6%) [19] , TaqMan (n = 19, 60%) [16] , [20] – [24] , and SYBR Green [23] (n = 1, 3%)-based real-time RT-PCR assays ( Table 1 ). As many as ten published protocols (indicated in Table 1 ) were used by participants and only three methods were established “in house” (9.37%). Eight laboratories used the TaqMan RT-PCR developed by Drosten et al. [20] , and six of them reported using the TaqMan RT-PCR developed by Bae et al. [21] , in both cases with varying performance depending on the reporting laboratory.
Performance varied among the 28 laboratories ( Table 1 ). Five out of 32 (16%) analyses reported met all criteria with optimal performance; 13 out of 32 (41%) test results achieved non-optimal results due to the failure to detect one or more YFV strains (in 12 sets of results wild-type strains were not detected, whereas in one data set the YF-17D strain was not detected). One laboratory did not report any positive results. Additionally, 78% (25 out of 32) of laboratory results reported false positives; 21 laboratory reports falsely identified YFV in samples containing other flaviviruses (#3 and #11) and four do so in negative samples ( Table 1 ).
Serial dilutions of YFV-17D (samples #2, #9, #12, #4, and #14) were used in order to test the sensitivity of the different methods. To estimate the effect of virus concentration on RT-PCR performance, Logit analysis was carried out using cumulative RT-PCR-positive results reported for each sample of the 10-fold YFV-17D dilution series ( Figure 1). The data demonstrated that 50% of positive performance could be expected at a concentration of 2.8×10 2 GE/sample (95% confidence interval [CI] 11.7–1.2×10 3 GE/sample). Ninety-five percent performance could be expected for 1.5×10 6 GE/sample (95% CI 1.9×10 5–2.8×10 8 GE/sample).
Data points represent individual samples in the panel. Thick line is the regression line calculated on the basis of a logit model (dose–response curve), and thin lines are 95% confidence intervals.
Comparison of the results obtained for similar concentrations of different YFV strains ( Table 1 ) also suggests the effect of virus type on the performance. Thus the results for YFV-17D were significantly better than those for the IvoryC1999 strain (McNemar's test was performed for sample #12 versus sample #13 and for sample #4 versus sample #1). The performance for YFV-17D was also significantly higher than that for the Brazilian strain (McNemar's test performed for sample #12 versus sample #10 and for sample #4 versus the sample #5). At the same time no essential difference in test performance was found when comparing Brazilian to Ivory Coast YFV samples.
Only four laboratories used two different molecular techniques for the evaluation, an advantageous approach to exclude false positives/negatives in routine diagnosis. The four laboratories used a combination of TaqMan RT-PCR and RT-nested PCR techniques. Laboratories no. 3 and 16 achieved better scores when using the TaqMan protocols, whereas laboratories no. 17 and 22 did with the RT-nested PCR ones ( Table 1 ).
Logit analysis was also carried out separately for results obtained with different RT-PCR techniques. For TaqMan-based real-time RT-PCR methods 50% performance could be expected when viral genome concentration was equal to 1.6×10 2 GE/sample (95% CI [0.1–584]) while for the RT nested PCR protocols it was achieved at 5×10 3 GE/sample (95% CI [87–3.7×10 4]). 95% of certainty was achieved with TaqMan real-time RT-PCR at 1.1×10 4 GE/sample (95% CI [3×10 3–2×10 7]), compared to RT nested PCR at 1×10 8 GE/sample (95% CI [2.4×10 6–2.8×10 15]), suggesting a better sensitivity of the TaqMan protocols. However, t-test comparing the TaqMan real-time methods' performance to that of RT nested PCR protocols did not reveal any significant difference in performance of the methods applied. In general, it seemed that the success of the analysis depended rather on the performance of the individual laboratories than of the format of the RT-PCR.
We requested further information on the number of copies of YFV genome in the samples sent to the participants in order to estimate the laboratories' experience in viral load determination. Only eight results out of 32 (25%) reported quantitative results (data not shown), although 20 laboratories reported results obtained by real-time-based procedures which are suitable to provide quantitative data. Among the reported results, five provided the quantification estimation as Ct values which give very limited data to accurately estimate the samples' viral load in the absence of calibrated standards.
Another point to consider in view of the results reported is the use of methods for the generic detection of flavivirus genome. Nine laboratories reported results using different pan-flavivirus protocols [18] – [20] , [23] , and five of them correctly differentiated the YFV samples from other flaviviruses in samples #3 and #11 by sequencing. This approach (detection plus sequencing) is suitable for diagnosis purposes as long as a good sensitivity is reached.
Some non-optimal results were due to the presence of false positives, mainly in sample #3 (only 25% of correct results) containing the four dengue serotypes and sample #11 containing other flaviviruses ( Table 1 ). Four laboratories reported false positive results in samples containing only human plasma, which indicates the need to improve laboratory procedures since carry-over contamination must be suspected.
Most of the serology laboratory results were obtained by using IIF for the detection of specific YFV antibodies (20 out of 31, 65%), but also ELISA (5 out of 31, 16%), seroneutralization (5 out of 31, 16%), and haemagglutination (1 set of results, 3%) assays.
Performance varied, depending mainly on the method used and the subclass of antibodies to be detected but also on the performing laboratory ( Table 2 ).
Four out of 25 reports (16%) using IIF or ELISA tests did not include results for IgM antibodies, whereas in the case of IgG detection only three laboratories (12%) did not report results, testing only the presence of IgM as marker of infection.
Of the 21 test results for IgM analysis, ten (48%) did not report the presence of anti-YFV-17D IgM antibodies even in the first serial dilution of YFV-17D-positive sera. Among them, six results out of ten (60%) were obtained using a commercial IIF test (EUROIMMUN), three out of ten (30%) using “in-house” ELISA, and one out of ten (10%) using a published IIF protocol [25] . In general, however, in the serial dilutions of samples containing anti-YFV antibodies against the South-American YFV strain, a good sensitivity profile was observed. Only seven out of 21 test results referring to IgM (33%) did not report anti-YF IgM antibodies in sample #1, the sera sample most diluted. Of these, two out seven (28.5%) laboratory results were obtained using an “in-house” ELISA test, two out of seven (28.5%) using an “in-house” IIF method, and three out of seven (42.5%) using a commercial IIF assay ( Table 2 ).
Two laboratories out of 25 (8%) performing IIF or ELISA assays reported false-positive IgM detection in sample #2 which contained antibodies against WNV. However, as many as four laboratories (16%) reported false-positive results in samples #8 and/or #12 (negative control samples).
Regarding anti-YFV IgG results reported during this EQA, a better performance was apparently achieved by laboratories using IIF protocols, as they obtained a higher score in the evaluation ( Table 2 ).
Two laboratories out of the 22 (9%) sending IgG results completely failed to detect IgG antibodies. No laboratory reported the presence of IgG antibodies against YFV (African origin) in samples #5 and #10. In the serial dilution containing anti-YFV antibodies against a South-American YFV strain used as sensitivity control, 86% of the data reported the presence of anti-YFV IgG antibodies in sample #14, 68% in sample #13, and 45.5% in sample #1. However, in eight test results (36%) the presence of anti-YFV IgG (17D) was not detected in the lowest serial dilution of the YFV-17D-positive sera. Among these, four laboratories out of eight (50%) used a commercial IIF assay (EUROIMMUN), two (25%) an “in-house” IIF test, one (12.5%) an IIF assay purchased from the Bernhard-Nocht-Institut (Hamburg), and one (12.5%) used two-in house ELISAs. In 13 reports (59%) IgG detection in YFV-17D samples failed for sample #11, and in 18 reports (82%) for sample #7 (highest dilution). Unexpectedly, none of the laboratories using anti-YFV ELISA detected the presence of IgG properly. Laboratory no. 19 reported the detection of IgG in samples #1 and #7 but not in previous dilutions of the same samples.
False IgG positives were reported in six laboratory results, three of them in samples #2 and #9 containing antibodies against other flaviviruses, and another three in sample #12, a negative control serum. The fact that other laboratories using the same commercial assays did not report these false positives results, suggests a problem in the operational procedures rather than a non-specific cross-reactivity of the assays.
In general, no difference in performance was found for IIF and ELISA assays or for the usage of commercial versus “in-house” assays (data not shown), nor was a difference found in the detection accuracy for IgM and IgG anti-YFV antibodies (McNemar's test).
The data obtained by seroneutralization assays do not allow to distinguish among IgM and IgG antibodies since the assay determines the presence of total neutralizing antibodies. Seroneutralization assays are expected to be the most specific ones in flavivirus serological diagnostics. In this study they showed a good performance with high scores ( Table 2 ). Indeed, even in the low-titre sample #5 (IgM−/IgG+) laboratories nos. 15 and 30 reported a correct positive result. However, the presence of false-positive results for samples #8 and #12 raises the question whether these results are due to a higher sensitivity or a strong background in the test. Control samples for the cytotoxicity effect and controls for virus infectivity must be included in each assay to be able to interpret the results correctly.
The haemagglutination assay which was only used by laboratory no. 20 showed a very low sensitivity, with only sample #14 reported as positive ( Table 2 ).
This is the first international EQA on YF diagnostics. The increasing importance of this disease in Africa and the Americas, and the risk of expansion to other areas, makes it necessary to assure that the methods used for YFV diagnostics and surveillance are working properly where they are already implemented.
Among the participating laboratories 20 out of 36 (56%) reported both serological and molecular results, indicating that they routinely include both approaches in their diagnostic algorithm. However, 22% (eight out of 36) of the participants applied only molecular or serological techniques, respectively, leaving room for the presence of false-negative results depending not only on the accuracy of the methods but also on the time period between the onset of disease and the diagnosis. We recommend combining both molecular and serological methods to provide the best accuracy in the diagnosis of wild-type YFV infections and YFVAE, an approach which increases the diagnosis window while minimizing the risk of false-negatives results. Likewise we also recommend the use of paired samples, when possible, to validate the results obtained in acute samples by confirming the presence/absence of seroconversion. This strategy is useful for the detection of false positives due to cross-contaminations in the case of molecular diagnosis or false isolated IgM positives in the case of serological diagnosis.
Regarding molecular diagnosis EQA, the participants using TaqMan real-time RT-PCR-based techniques overall showed a better performance with a higher sensitivity than other assays. However, the major limitation for the implementation of these assays is the costs of both thermocyclers and reagents which hamper a generalized application in the field.
One of the main weaknesses observed during this EQA was the inability of some protocols to detect the YFV genome of wild-type strains. Five out of eight laboratories which failed to recognize all wild-type YFV strains used the TaqMan real-time RT-PCR described by Bae et al. [21] , two out of eight used “in-house” methods, and one applied the method previously described by Brown et al. [22] who already pointed out that false negatives might occur when using this protocol. One laboratory used a TaqMan real-time RT-PCR previously described exclusively for the detection of YFV vaccine strain 17D [16] , which would explain the results obtained. However, the methods mentioned above showed a very good sensitivity profile against YFV-17D, making them useful tools to identify viral genome in suspected YFVAE or research, but obviously not to identify suspected wild-type cases. The presence of mismatches between the oligonucleotide and the viral target sequences might explain the failure in genome amplification. This denotes the need to adapt and up-date regularly oligonucleotide sequences in use in diagnosis laboratories to detect the presence of mutations in the circulating strains which may compromise the ability of the assay to amplify and/or detect the targeted sequence leading to false negative results. However, among those laboratories which failed to detect only one of the wild-type strains (South American [Brazil] or West African [IvoryC1999]), two laboratories reported the use of the TaqMan real-time RT-PCR described by Drosten et al. [20] , one laboratory used the RT-nested PCR by Kuno [15] , and one laboratory used an “in-house” protocol. Other participants applied the method published by Drosten et al., with optimal detection of all YFV strains. Therefore, it seems in these cases that failure in detecting some strains might more likely be due to the specific performance of the laboratories rather than to the techniques themselves, suggesting the need to revise the running protocols of these laboratories to improve the quality of their results.
One laboratory completely failed in the detection of all positive samples. It can not be excluded that the gamma irradiation of the samples for inactivation could have resulted in nicked RNA that would affect those methods amplifying fragments of around 600 bp [26] as it is the case of the method used by this laboratory [17] . However, other laboratories reported good results with bigger amplification targets so probably other factors might have influenced the performance of this laboratory.
Similarly, the presence of false-positive results in flavivirus RNA-free samples also denoted the need to optimize laboratory practices in order to avoid the occurrence of cross-contamination.
It is remarkable that for the sample containing genome of the four dengue serotypes (sample #3) only 25% of the results were correct. In the meantime the possibility of the presence of contaminant traces of YFV genome due to the processing and preparation of the samples has been excluded, and these results could indicate some degree of non-specificity in the techniques. As dengue is a common differential diagnosis for YFV, such specificity issues should be taken into consideration while interpreting dengue or YFV diagnostic testing results.
Twenty-eight percent of the tests reported used a generic flavivirus assay for the detection of YFV RNA, and 55.5% of these correctly differentiated the presence of YFV genome from that of other flaviviruses by consecutive sequencing. This approach might be advantageous for diagnostic purposes in laboratories which need to cover the possibility of infections with different flaviviruses without holding available a specific protocol for each of them. However, we must point out that the use of these generic methods must always be accompanied by sequence identification, mainly in endemic areas where usually more than one of the flaviviruses circulates.
As in previous EQA studies, data regarding viral load were scarce even though the use of real-time techniques was prominent [12] .
Regarding the serological results of this EQA, one of the main observations was the fact that 16% of the participating laboratories do not routinely include the analysis of specific anti-YFV IgM in their diagnostic algorithms. It is well known that for the serological diagnosis of an acute YFV infection it is preferable to test for the presence of IgM which appears for a short period of time soon after the infection, confirming a recent contact with the virus. The presence of IgG does not provide a good proof of recent YFV infection and requires the analysis of a second sample to confirm a rise in the antibody titres. In general, the sole detection of IgG would only confirm a previous contact with YFV, even by vaccination, or with another flavivirus as serological cross-reactivity is more pronounced for IgG detection than for IgM, as suggested by this EQA and previous reports [27] – [29] . It is important to note that in general the existence of this cross-reactivity is the major weakness of the serological diagnosis, not only for YFV but for all flaviviruses. Seroneutralization assays are considered the “gold standard” for specific identification of a positive immune response. However, some degree of non-specificity can not be excluded and such a technique requires the use of well-characterized controls. Additionally, seroneutralization assays can perform poorly in samples with multiple/subsequent infections unless multiple samples are available. Also the material, the expertise, and the time required to perform a seroneutralization assay do not make it a proper choice for early diagnosis.
Fortunately, from the results of this EQA it has become obvious that false positives due to cross-reactivity are not the main limitation of the serological techniques used by the participating laboratories, and that most of the specificity difficulties could be solved by proper standardization of the protocols and the use of adequate controls during the assays.
One of the main issues that deserve more attention is the apparent lack of sensitivity regarding anti-YFV-17D IgM detection in 48% of the results reported, while a good sensitivity profile was observed in wild-type infection sera. This should be taken into account when assessing the protection provided by the YFV vaccines by determining IgM levels.
In the case of IgG detection, apparently the quality of the results depends mostly on the performing laboratories since their results differ even when using the same technique. The lack of detection of IgG in sample #5 has no major impact on the conclusions of this EQA since the samples from West African wild type infection contained the lower titre of antibodies compared to the others, and required very sensitive techniques for detection, which could only be achieved by seroneutralization assays in this EQA. Similarly, the presence of false-positive results in those laboratories with other correct results makes it unclear whether a low threshold of detection could imply a higher risk for false-positive results to occur.
We conclude that the main differences in the molecular diagnosis results might be more related to the handling of assays and specimens, pointing out the importance of regularly revising and improving the operational protocols. The different profiles of strain detection obtained with each protocol must be taken into account by the laboratories which must select those protocols most suitable for their particular diagnosis purpose (wild-type infections vs. YFVAE).
In general, commercial serological assays showed a very good sensitivity profile in this study for both IgM and IgG detection of wild-type origin, but not for antibody detection against the vaccine strain YFV-17D. Commercial serological assays used for diagnostic purposes show a better specificity when compared to “in-house” ones. However, their use raises some concern when evaluating the immune response elicited after vaccination.
No difference in the sensitivity detection of IgM and IgG anti-YF antibodies has been found in this EQA, in contrast with the results observed in previous EQA studies on other arboviral infections [11] , [30] , [31] . However, the different titres of IgM and IgG in the samples included in this EQA make this comparison quite general.
The low participation of endemic countries in this EQA, even though widely announced, points out the need to encourage more laboratories to implement YFV diagnosis techniques and to participate routinely in quality assurance programmes.
The authors are grateful to J. Kraft for excellent assistance during the EQA activity and to U. Erikli for copy-editing.
Competing Interests: The authors have declared that no competing interests exist.
Funding: This study was performed by the European Network for the Diagnostics of ‘Imported’ Viral Diseases-Collaborative Laboratory Response Network (ENIVD-CLRN) funded by the European Centre for Disease Prevention and Control (ECDC), contract no. ECDC/2008/011. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.


CUTTHROAT ISLAND A film review by Craig Good Copyright 1996 Craig Good
So Jeff and I got to the crummy local 15-plex just a few minutes too late to see "Sense and Sensibility" on Saturday. The logical alternative was to see "Cutthroat Island" instead. Right? It turned out not to be such a bad choice.
I'm not going to try to tell you that this is great cinema. It's not going to make many top ten lists, and even this year I don't see it grabbing a nomination for Best Picture of the Year. But was it fun? Yup.
>From the first stunt set piece, where Geena Davis the pirate (if you can't suspend that particular disbelief long enough for her to convince you, don't bother going any further) plunges toward the ocean floor to cut her father loose from the anchor which is pulling them both below the waves, I began to make mental comparisons to "Goldeneye". I thought that if that incomprehensibly dull Bond film had had one or two gags that good, it would not have been such a disappointment. But the scale and outrageousness of the gags in "Cutthroat Island" do nothing but build from there, all the way to the explosive finale.
This is a pirate film which aims to please and, for the most part, delivers. Nobody said "Arrrgh, Matey" nor "keelhaul", but peg legs, plank-walking, cannon fire and sabre duels are delivered by the galleon. It may be obvious to point out that this is not a finely-crafted character piece. Director Renny Harlin instead paints with broad, bold strokes which are more often than not perfectly appropriate. I mean, why have a boring establishing shot when you have a good helicopter camera rig close at hand?
The dialogue doesn't so much sparkle as fizzle most of the time, with occasional and worthwhile exceptions where it blazes. But the action, gorgeous locations, and and production design deliver in such outrageous proportions that it is possible to forgive many weaknesses. Were I a pyro/FX man on this picture, I would have left the set every night saying, "Man, I love my job". It's not just scale which makes it work, although heaven knows this is huge, but the playful imagination which went into nearly every setup. Call me perverse, but I found poetry in the way giant cannons were catapulted through the air during the point-blank sea battle. And Geena Davis' stunts (aided, no doubt, by digital wire removal) were often *literally* over the top.
If you found this year's Bond flaccid and still hunger for a fun romp, perhaps the turgor of Geena Davis will sate that appetite. It's no "Citizen Kane", but in the end, "Cutthroat Island" delivered everything I wanted but never got from "Goldeneye".
--Craig<EMAIL_ADDRESS>Toy Story: $125.5 Million and counting...
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DISCLAIMER:
Everything I say in this report is maybe a million times under exaggerated. The feeling of a salvia trip is beyond words, the state of consciousness I entered is literally the opposite of what my rational mind will have me believe is reality, therefore there are really no words that can properly describe a salvia trip. But there’s one that comes pretty damn close: chaos.
THE SET:
I had been experimenting with various psychedelic drugs for a couple weeks before this trip. I had done Salvia twice before, THC (Cannabis), Psilocybin (Magic Mushrooms), LSA (Morning Glory Heavenly Blue Seeds) and I had been experimenting with both lucid dreaming and sleep deprivation. All my experiences with psychedelic “tripping” had been completely positive and I had become generally used to the psychedelic feeling of mind expansion to the point of where I could function almost completely normally when on a substance. My prior experiences with salvia had been generally light, especially when compared to the following trip, and euphoric with closed-eye visuals and an intense body high, an almost drunk feeling with an obvious presence of DXM style dissociation.
Everything in my life was pretty happy, I was happy with my self and the people I associated myself with. I had been having mild mood swings the week prior but nothing completely out of the ordinary was bothering me. I had also smoked marijuana and sniffed 30 mg of Dexedrine and around 30 mg of a chemical compound similar to Dexedrine but effective in the dopamine release in your brain.
THE SETTING:
It was a cloudy day, not my ideal day for a psychedelic trip (I find the sunnier the better). We were in a small kind of clearing of nice, fresh grass with bushes on either side of us. It was between 1:45 and 2:30 that all the trips happened, mine being at around 2:00 – 2:10. There were seven of us in total, a good group of friends I like to explore drugs with. An eighth person would come slightly later, after the main part of my trip was over.
THE DOSE:
We agreed on all taking one giant hit of the 20x extract from a small, glass pipe and holding it in for as long as we could before exhaling and having the following breath as another hit.
THE TRIP:
I watched the pipe get passed to four people before me. The first two didn’t seem to have any visual effects, both saying they only felt really baked or really drunk with a touch of the giggles and a strange bodily sensation. The next two seemed to get better hoots, the first just closed her eyes and kind of went off while the second just looked at his shoes and laughed really hard. The pipe then came to me. I took a giant hit and held it in for about thirty seconds before taking a smaller one and holding it in for another thirty seconds. This is when I started to feel it coming on, strange pressures on my body (particularly my legs) and abstract thoughts starting to formulate in my mind.
It is impossible to truly describe what happened next. The feeling of the drug kicking in was like falling asleep and awaking millions of years later in a completely different state of mind. The setting changed so much in mind as I blew out the smoke that it’s as if the events in the day that had taken place before had happened years ago, or had never happened at all. At first I forgot that I had taken anything. I put my head down and simply thought that I was baked, but when I looked up it struck me that this wasn’t the case.
The lighting was similar to that of twilight, and everything I looked at seemed to be facing a setting sun, my friends’ skin looked reflective and red while the air around everyone looked orange. I felt warm and comfortable as the body high came on, a relaxing, blissful feeling of nothingness and at the same time an electric shock that contains all time and matter. My mind seemed to split into two separate entities within my head: The Goddess, a distinctly feminine presence that allowed me to look at nature and people and mentally touch them. I’d identify this as the spirit of Salvia. And The Scientist, a whole other presence that allowed me to articulate abstract thoughts that seemed at once both infinitely true and comically false. I now recognize this as my mind coping with the effects of the drug.
The Scientist was focused on a constant thought that was running through my mind: I am a house. I could feel the rooms inside of me and inside each room were different people I knew. I could walk amongst these rooms, enjoying everything inside them and talking to the people within them. I still hadn’t realized I had done a drug, and this had all happened in maybe ten seconds. The next ten seconds have continued to baffle me no matter how many times I try to revisit and understand them.
I looked up and came immediately to the realization that I was on some kind of drug. My mind registered that I had done salvia but I still couldn’t quite comprehend why, when or how. The lighting had changed from twilight to fractal combinations of colors I hadn’t ever seen before, colors I can’t describe because they have absolutely no relation to any colors the human spectrum of light can normally perceive. Everything natural around me was immediately “touched” by the finger of the Goddess, giving me a sense of total understanding. The Scientist’s thoughts about me as a house were confirmed as I realized all my friends were merely houses and we were all part of a cul de sac in a neighborhood.
This is where I stopped all kinds of resistance and stopped letting myself have a trip, I let the trip have me. I looked directly opposite myself and there sat one of me friends, C, smiling back at me. This comforted me and I could feel The Goddess smile but at the same time I could feel The Scientist burst into a panic attack as he saw that C was both in one of the rooms inside my body and outside of it looking at me. I broke into hysterics.
There’s a short blackout here, I don’t know exactly what happened but I had wandered over to one of the bushes one either side of the clearing, and I was staring at the fluorescent green of the leaves. I could see them breathing and could feel their life force illuminating me as I placed a leave on my hand. This gave me an extreme appreciation for nature, The Goddess was trying to help me communicate with the plant. My thoughts were interrupted by laughter from my friends all around me.
I looked back over my shoulder to see my friend, C, who had just taken his dose. He was squirming around on the ground as two of my other friends tried to keep him in place. He later explained that he had thought the ground was in fact a vertical cliff, and he was trying to climb it as everyone weighed him down. I continued to watch him, The Goddess laughing and enjoying herself, The Scientist taking notes and analyzing my setting, and found his tripping a comfortable relay of my experience. I went over to stand by him as he laughed as hard as he could, the thought of this broke me into laughter.
This is where the effects started to diminish themselves. I started to feel like I was on the cusp of something extraordinary and that there was a lot to learn from the Goddess, but the Scientist began to take over, leaving the visual effects lesser pronounced as the bodily high calmed and my thoughts resumed a linear format. At this moment I swear I actually heard the Goddess whisper “goodbye” into my ear. I have no doubt the sound was actually there – audio hallucinations aren’t this real. I still felt mild tripping, the ground was still breathing and the confusion that accompanies the aftermath of a salvia trip (a different confusion then the actual trip, but similar to the feeling of confusion on Magic Mushrooms) was starting to wrap itself around my mind. I laid back and let my thoughts attempt to organize themselves, but all my brain was doing was trying to forget what had happened.
My theory for why the brain tries to dispose of the trip memories is that the logical part of my brain realizes that if the information is stored, there is a chance that it could drive me into thinking in ways the brain isn’t programmed to thinking in, the ways it should be thinking in, but has been neglected from doing so by the relentless destruction of the true mind by society. So the brain forgets as much of the trip as possible, that is unless you really try to remember it and focus on the meaning behind it.
So I just chilled, let myself come down off the drug as a couple of my other friends smoked their share. I talked with my friends about some trippy thoughts, we all understood that none of us really wanted to share our trips yet. Another one of my friends showed up around 2:20 and had one hit from the pipe. He looked particularly tripped out but he looked like he wasn’t really enjoying himself. One thing that he said during his trip that really proved to me that salvia is not quite a drug, more a spirit that one takes on, was “Dude, you look like a house…” In my opinion, the fact that I had hallucinated myself as a house earlier was no coincidence, we had both been given a piece of the same blessing from The Goddess, salvia.
THE AFTERMATH:
I didn’t reach baseline for awhile. Especially not in a mental sense, my mind was still in complete disbelief of what it had experienced, it is certainly a hard task to try to comprehend that kind of trip with a sober mind. My bus ride home was enjoyable, I listened to a couple of artists on my iPod before coming to The Jimi Hendrix Experience. The music, the lyrics – pretty much everything about the Jimi Hendrix Experience seemed completely real and natural. The acoustic songs I listened to sounded like the voice of an old forest, secrets being whispered between clouds.  The music put me in a really good mood and the feeling of the bus’ movement was calming.
OVERALL:
Easily the most meaningful trip of my life and very enjoyable one as well. I still have a lot to thing about in terms of what exactly the Goddess was trying to communicate with me, and I hope to visit her again.
Experience Reports are the writings and opinions of the individual authors who submit them.
Some of the activities described are dangerous and/or illegal and none are recommended by Erowid.


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Autopad extends the input data set (of type VectorType) with zeros to make the total number of data items equal to the nearest larger power of two. Autopad has no parameters in order to make it convenient to use with the FFT unit in order to make the FFT run most efficiently. If the input set is already a power of 2 it is not extended.
The way that this unit acts on VectorType inputs varies according to the type. A pure VectorType will be extended as described above. If the data are irregularly sampled, they will be extended with a regular spacing equal to the size of the first or last data interval, according to whether they are placed at the beginning or the end. A SampleSet or a ComplexSampleSet will be extended as described above and its sampling rate will not be changed. A Spectrum or ComplexSpectrum will be extended as described above; its frequency resolution will not be changed, and the parameter nFull (the number of points in the full data set from which it might have been derived by a Fourier transform) is increased to the new size. If the spectral data are narrow-band, the new length of the extended set is interpreted as the size of nFull, not the size of the bandwidth of the data held in the object. Note that Spectrum and ComplexSpectrum data sets cannot be extended at the front, since it does not make sense to add points below zero frequency. More details are given in the documentation for these spectral types. If the input set is a Histogram, then the data set will be extended as described above. If the zeros are to be added at the beginning and the first delimiter is negative infinity, then nothing is done; similarly if the zeros are to be added at the end and the last delimiter is positive infinity, nothing is done.


--- layout: page title: Demonstrated Scenario permalink: 00-timeoff-demo.html ---
Browsing to timeoff-management application URL presents a login window:
timeoff-management
In this window, click Register new company


Faulty loop-line caused derailment of train in Sirajganj: probe
by bdnews24.comAn investigation into the derailment of a train in Sirajganj's Ullapara has blamed a flaw in the tracks for the crash.
Sirajganj's Additional Deputy Commissioner Feroz Mahmud, convenor of the probe panel, submitted his findings to Deputy Commissioner Faruk Ahammed on Thursday.
On Nov 14, a fire engulfed the power car and engine of a Rangpur Express train as it went off the tracks near the Ullapara Railway Station.
The probe panel in its 29-page report drew up five recommendations while identifying the faults in the loop line as the reason for the accident, said the deputy commissioner.
“It said that the stock rail and tongue rail at the scene had to be locked, but they weren’t. There was a gap between the two lines. That’s why the engine was derailed when it reached the spot.”
The deputy commissioner refused to go into the details of the report. The administration will reveal the details after forwarding the report to the ministry, he said.


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In the blink of an eye, Google Summer of Code (GSoC) 2018 has come to an end. During the three months long coding period, I have contributed several patches in VOC repository of BeeWare, all working towards the ultimate end goal of running asyncio module in VOC. In this blog post (which is my first actual blog post by the way 😄), I will document what I have done so far, why I couldn't make it to the end goal (yea, unfortunately I couldn't get asyncio to work at the end of GSoC 2018), and what's left that needs to be done in order to achieve the end goal (or at least make part of asyncio work).
Building Foundation
The first error that the transpiler throws when attempting to compile asyncio module was "No handler for YieldFrom", so it makes sense to start from this issue first.
Another feature related to generator was Yield expression. Before GSoC 2018, Yield statement in VOC was just a statement, meaning yield could not be used as expression. Generator methods such as generator.send, generator.throw and generator.close were not supported as well. Those features are what make asynchronous programming with generator possible, so I spent a few weeks to extend generator functionality in VOC, laying down the path to asyncio module.
PRs related to generator are listed below:
Nonlocal Statement
Nonlocal statement was another syntax not supported by VOC. After completion of generator's features, implementing this is the next step towards compiling asyncio module.
Implementing this feature took about 3 ~ 4 weeks as this is not as trivial as it seems. I took several approaches on this, while some of them do work, the code is not pretty and hacky, which could come back to bite me/other contributors in the long run. After many discussions with Russell, I refactored the closure mechanism in VOC and took a much cleaner approach in nonlocal implementations. I must admit that I took some short-cuts for the sake of "making nonlocal works" in the process of implementing nonlocal statement, resulting in poor design and messy codes. Many thanks to Russell, who helped me to improve my coding style and told me not to be discouraged when I'm stuck. 😄
Related PRs:
The Collections Module
Next item on my hit list was pure Java implementations of the collections module. asyncio module depends on 3 data structures from collections, namely defauldict, Deque and OrderedDict. Two of them (defaultdict and Deque) are implemented in C in CPython, plus they have good analog in Java, so it makes senses to implement the module in Java. Porting defauldict, Deque and OrderedDict to Java in VOC is relatively straight-forward, taking about 1.5 weeks to complete.
Related PRs:
Other PRs submitted during GSoC 2018
- PR #817 : Added coroutine related exception class [WIP] (closed due to not needed)
- PR #836 : Changed Bool construction to use getBool instead (merged)
- PR #847 : Add custom exceptions test cases (closed due to more comprehensive handling in PR #844)
- PR #849 : Fixed Unknown constant type <class 'frozenset'> in function definition (merged)
- PR #858 : Added test case for Issue #857 (merged)
- PR #860 : Added test case for Issue #859 (merged)
- PR #862 : Added test case for Issue #861 (merged)
- PR #867 : Fixed Issue #866 RunTimeError when generator is nested in more than 1 level of function definition (merged)
- PR #868 : Fixed Issue #861 Redefining nested function from other function overrides original nested function (merged)
- PR #879 : Fixed Incompatible Stack Height caused by expression statement (merged)
- PR #901 : Added test case for Issue #900 (merged)
- PR #788 : Implements asyncio.coroutines [WIP] (open, the dream 😎)
Issues submitted during GSoC 2018
- Issue #861 : Redefining nested function from other function overrides original nested function (fixed in PR #868)
- Issue #866 : RunTimeError when generator is nested in more than 1 level of function definition (fixed in PR #867)
- Issue #828 : Finally block of generator is not executed during garbage collection (open)
- Issue #857 : Complex datatype in set cause java.lang.StackOverflowError (open)
- Issue #859 : Duplicated values of equivalent but different data types in set (open)
- Issue #900 : Exception in nested try-catch suite is 'leaked' to another enclosing try-catch suite (open)
- Issue #827 : Maps reserved Java keywords to Python built-in function/method call (closed)
Towards The Ultimate End Goal
Unfortunately, three months of GSoC coding period was not enough for me to bring asyncio module to VOC. The nonlocal statement implementation was the biggest blocker for me mainly because I didn't think thoroughly before writing code. If I were to plan carefully and lay out a general coding direction, I would've completed it in much shorter time and have time for other implementations. An advice for the aspiring and upcoming GSoC-er, don't rush your code, make sure you know 100% about what you're doing before diving into the codes.
With that said, following are the list of modules to be implemented/ported to Java before asyncio will work in VOC:
- socket module (a bit tricky since Java doesn't support Unix domain socket natively)
- selectors module (high level I/O operations)
- threading module (might be easier to implement this first since threading in Python is an emulation of Java's Thread)
- time module (partially implemented in VOC)
Final Thoughts
Huge thanks to my mentor, Russell Keith-Magee for accepting my proposal, providing guidance and encouraging me when things didn't go as intended. It is truly an honor to be a part of the BeeWare community. I had a blast contributing to BeeWare project, and I'm sure I will stick around as a regular contributor. Also shout out to the BeeWare community for answering my queries and reviewing my pull requests. 😄
- الإدخال التالي
- 2018 Google Summer of Code - VOC Optimization
- الإدخال السابق
- Project Spotlight: Colosseum


DC 1st Look: SUPERMAN Tries to Convince JIMMY OLSEN He's Clark ... Jimmy Isn't Buying
by Newsarama StaffThursday it was an image, and now Friday DC released several pages from next month's Superman #18 by Ivan Reis, Joe Prado and Alex Sinclair. The issue is written by Brian Bendis.
Superman #18 ispart one of the "The Truth," the storyline that reveals Superman's secret identity to the world that may or may not be due to the machinations of Leviathan.
Check out the five new pages in our gallery, which seem to show Superman trying to convince Jimmy Olsen, he's Clark Kent ... unsuccessfully.
"The secret is out!" reads DC description of the issue. "In a story already making headlines, Superman's family is scattered across the galaxy, and our hero must confront some of his greatest concerns about himself and his place in the galaxy. Legendary artist Ivan Reis joins writer Brian Michael Bendis as they introduce the biggest change in the Man of Steel's life ever!"
Superman #18
Written by Brian Michael Bendis
Cover and Interior art by Ivan Reis and Joe Prado
Colors by Alex Sinclair
In Shops: Dec 11, 2019
Final Orders Due: Nov 18, 2019
SRP: $3.99


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Module BsCron.CronJob
Module for creating new cron jobs
-
val make : [ `CronString of string | `JsDate of Js.Date.t ] -> ((unit -> unit) -> unit) -> ?onComplete:(unit -> unit) -> ?start:bool -> ?timezone:string -> ?context:< .. > Js.t -> ?runOnInit:bool -> ?utcOffset:int -> ?unrefTimeout:bool -> unit -> t
-
make(cronTime, onTickFunction)
returns a cron job that when started will run on the schedule specified by the cron time.- parameter cronTime
-
The syntax of
cronTime
can be in cron format (e.g.2 * * * * * *
) or as aJs
.Date.t.
- parameter onTick
-
The function that will be run for every job execution (specified by the cron time). It will be passed the function given as
onComplete
parameter as it's parameter.
- parameter onComplete
-
A function that will fire when the job is stopped with
stop
, and may also be called byonTick
at the end of each run (it is passed toonTick
).
- parameter start
-
Specifies whether to start the job just before exiting the constructor. By default this is set to false. If left at default you will need to call
start
in order to start the job (assuming job is the variable you set the cronjob to). This does not immediately fire youronTick
function, it just gives you more control over the behavior of your jobs.
- parameter timezone
-
Specify the timezone for the execution. This will modify the actual time relative to your timezone. If the timezone is invalid, an error is thrown. You can check all timezones available at Moment Timezone website. Probably don't use both
timezone
andutcOffset
together or weird things may happen.
- parameter context
-
The context within which to execute the
onTick
method. This defaults to the cronjob itself allowing you to callthis.stop()
(this is for JS, uncertain how this is in ReasonML/OCaml). However, if you change this you'll have access to the functions and values within your context object.
- parameter runOnInit
-
This will immediately fire your
onTick
function as soon as the requisite initialization has happened. This option is set tofalse
by default for backwards compatibility.
- parameter utcOffset
-
Set an UTC offset for your timezone instead of using the
timezone
parameter. This is for a Moment-object, so see Moment-documentation for more info. Do not useutcOffset
andtimezone
parameter together.
- parameter unrefTimeout
-
If you have code that keeps the event loop running and want to stop the node process when that finishes regardless of the state of your cronjob, you can do so making use of this parameter. This is off by default and cron will run as if it needs to control the event loop. For more information take a look at timers#timers_timeout_unref from the NodeJS docs.


--- layout: post title: Examples of Institutional Failure date: 2014-04-24 19:47:29.000000000 -05:00 type: post published: true status: publish categories: [feed] tags: takeoff meta: _publicize_pending: '1' _edit_last: '8005175' author: login: dvollrath email<EMAIL_ADDRESS>display_name: dvollrath first_name: '' last_name: '' ---
Miles Kimball posted a link this article from the WSJ on the mess that is Nigeria's electricity grid. A key factoid is that Nigeria produces as much electricity as Montana, yet Nigeria has about 170 million people while Montana has only 1 million. The author (Drew Hinshaw) has comparisons for other African countries that are as disturbing.
The story focuses on Tony Elumelu, who recently purchased a power plant. His logic here is sound:
Thanks to all-day outages, Nigerians consume scant electricity—less than Puerto Rico. Once electricity flows into their homes, though, tens of millions of people will rush to buy refrigerators, air conditioners, electric kettles, he added—all pulling power from his turbines.
But the real question is whether the institutions in Nigeria will allow this to happen. As mentioned in the article, there is rampant theft of electricity from the grid, the grid itself is old and failing, and money is often extorted from providers in exchange for not blowing their equipment up.
I don't know how to properly define "institutions", but when we say that institutions matter, I think this is what we mean. The gains to having regular electricity are obvious, but the question is whether someone like Tony Elumelu will be able to keep enough of the profits from selling electricity to make him stick with his investment.
In a completely unrelated, but to me equally shocking story, Miles Kimball was my professor for Money and Banking at Michigan - 23 years ago.


Alejandro Buitimea
Project Portfolio
Alex is a third year, Computer Engineering Major attending the University of California, San Diego.
Projects
SHPE Management Application
CSE 170 Application
Sleeping
...


The TMReadDefaultPort API call reads the default 1-Wire port number
and type from the system registry. The port number and port type are both
integers that describe on which port the 1-Wire network is connected and through
which port adapter type.
The port numbers can be an integer from 0 to 16. Most valid port numbers on PCs are 1, 2, or 3. The following is a list of port adapter types:
1 Older DS9097E-type serial port adapters
2 Parallel port adapters
5 Standard DS90907U-type serial port adapters
6 USB port adapters
For a description of the above types, see the TMEX Type Descriptions.
Parameters
portnum
Specifies the port number of the specific port (serial, parallel, or usb) where the 1-Wire network is located. Most port numbers on PCs are 1, 2 or 3.
porttype
Specifies the port adapter type. There are a variety of PC-to-1-Wire adapters available. For a short list, see above. For a more in-depth list of adapters and their respective port type numbers, please see the TMEX API Overview. Currently used numbers are: 1, 2, 5, and 6.
Return Value
1 =>
defaults were returned
-2 =>
defaults not found
See Also


👮 Welcome to the Jo🅱hu🅱 Authentication Microservice 👮
Please consult our
repo
for endpoint information.


About Us
Salmon Cookies are a delicious, high protein treat!  We have perfected the perfect salmon cookie based on our grandmother's recipe. Years ago, she used to bake a batch of these and send them off with us on our fishing expeditions.
We have improved upon her recipe by adding chocolate chips and making them dairy-free. They are full of protein (18 grams per cookie) and are rich in Omega-3 acids that are good for your brain.
They are delicious with a cup of coffee or as a nutritious snack by themselves.
Enjoy your cookies from any one of our 5 worldwide locations listed below.
We also do catering for special events and can ship to your location, so call for more information.
- OUR LOCATIONS
- United States:
Salmon Cookies Flagship
123 NW Market St.
Seattle, WA 98107 - Tokyo:
1-2-3 Higashi Shimbashi
Minato Ku Tokyo
105-7123 Japan - Dubai:
404 Al Mina Rd.
Bur Dubai
United Arab Emirates - Paris:
111 Boulevard Saint-Germain
75006 Paris
France - Lima:
Ca Peru 785
23578
Lima
OUR ONLINE STORE
The following items can be shipped to you!
Domestic: 3-5 buisness days International: please allow up to 2 weeks
Please call us to place your order-
Cookie Cutter $5 U.S. -
Custom Frosted Cookies $3 U.S. each -
T-shirt $15 U.S. -
Salmon Poster $12 U.S.


DECEMBER 28 The Holy
Innocents, Martyrs
FIRST
A reading from the first letter of St John 1:5-2:2
Beloved: This is the
message we have heard from Jesus Christ and proclaim to you, that God is light
and in him is no darkness at all. If we say we have fellowship with him while
we walk in darkness, we lie and do not live according to the truth; but if we
walk in the light, as he is in the light, we have fellowship with one another,
and the blood of Jesus his Son cleanses us from all sin. If we say we have no
sin, we deceive ourselves, and the truth is not in us. If we confess our sins,
he is faithful and just, and will forgive our sins and cleanse us from all
unrighteousness. If we say we have not sinned, we make him a liar, and his word
is not in us. My little children, I am writing this to you so that you may not
sin; but if any one does sin, we have an advocate with the Father, Jesus Christ
the righteous; and he is the expiation for our sins, and not for ours only but
also for the sins of the whole world.
The word of the Lord
RESPONSORIAL PSALM
Psalm 124
R. Our life, like a bird, has escaped from the snare of the fowler.
If the LORD had not
been on our side when men rose against us, then would they have swallowed us
alive when their anger was kindled.
R. Our life, like a bird,
has escaped from the snare of the fowler.
Then would the
waters have engulfed us, the torrent gone over us; over our head would have
swept the raging waters.
R. Our life, like a bird,
has escaped from the snare of the fowler.
Indeed, the snare
has been broken, and we have escaped. Our help is in the name of the LORD, who
made heaven and earth.
R. Our life, like a bird,
has escaped from the snare of the fowler.
ALLELUIA
R. Alleluia.  R. Alleluia.
GOSPEL
A reading from the holy Gospel according to Matthew 2:13-18
When the Wise Men
had departed, behold, an angel of the Lord appeared to Joseph in a dream and
said, "Rise, take the child and his mother, and flee to
The Gospel of the Lord


Lets say you need to validate an input only on blur
, but once
that results invalid you want to give your user feedback on validity as soon,
as he corrects the input, without waiting for 'blur' to happen again. Make that
a click of a "save" button and resulting $beforeSave
event instead of blur,
and it is definitely a good idea to mark each field as valid as soon as it is corrected.
Not to mention, that you can disable the "save" button untill $beforeSave
event
is valid for each input again.
_excludedNonValids
allows you to do jsut that by changing the behaviour of events that resulted
in invalid the last time they were validating, but would not noramlly participate in the current valiation.
The first input of this example showcases a typical case - maxLength(5) will be validated on blur
and if it result in invalid, it will validate on each symbol entered, untill length of the input becomes 5 or less,
at which point it will instantly become valid and won't trigger again untill next blur.
Overall avaliable options are:
"validate"
- the default behvaiour described above
"setValid"
- all events that do not participate in a triggered validation will
be set valid. Seccond input in the example showcases this. If you enter 7 symbols into it,
blur it and then enter another - blur event will instantly become valid. It will reevaluate on next actual
blur event as a part of normal workflow.
"doNothing"
- validation is only triggered for events that participate, others are left
valid or invalid as is, without recheck.
This behaviour applies to any validation, no matter what actually triggered it - be it the default
event,
a JS event or VALIDATION_REQUEST angular event.


A sample application that demonstrates how to manually query the system contacts provider using
the new ContactsContract
API, as
well as manually insert contacts into a specific account.
In most cases, you will want to ask the existing Contacts activity to handle these tasks for you, resulting in a more consistent user experience.


My Team
Jared
Manager
Id: 1
Email:
<EMAIL_ADDRESS>Office Number: 1
Alec
Engineer
Id: 2
Email:
<EMAIL_ADDRESS>GitHub:
ibealec
Grace
Engineer
Id: 3
Email:
<EMAIL_ADDRESS>GitHub:
gchoi2u
John
Intern
Id: 4
Email:
<EMAIL_ADDRESS>School: 2University


---
layout: post
title: Made In Madison Radio
date: '2005-07-22T01:57:00.000-05:00'
author: Tom Ray
tags:
modified_time: '2005-07-22T02:12:58.773-05:00'
blogger_id: tag:blogger.com,1999:blog-11319466.post-112201628603825824
blogger_orig_url: http://www.lorenzosmusic.com/2005/07/made-in-madison-radio.html
---
We have recently been added to the MusicMadeInMadison.com internet music radio playlist where you can hear a bunch of local artist in a streaming radio show. Check it out.


Where is your quiet place
to focus on, and Why?
I find that the bathroom mirror is a powerful tool for self-reflection.
The blue and white tones remind me something that resonates with peacefulness.
I love the wind and fresh air when there’s no cars or construction.
I find quiet places bothersome...if I’m in those places I have upbeat music playing.
My quiet place is my bed because it’s warm and soft.
I lost in my thoughts, with blankets on.
I tend to view my bed as a place to reflect myself and my worries.
It's a space that only belongs to me and no one else.
I really like the interior, which feels cozy.
It's decorated with all my favorite things and I can catch a break from the hectic city.
I have plants and love to see their growth and water them.
The changing scenery around makes me feel relaxed and happy.
I can feel the breath of nature interacting with me.
These landscapes allow me to examine my own changes as time flies.
It’s nice to have a place where you can escape from other people.
No one interrupts me while I'm focusing on art pieces.
...when I am showering it is the only place where I have no external distractions.
...the whole journey of going there is like a quick vacation.
I don't do any school work there, and it is my quiet place to focus on myself.
...being surrounded by dense forest has lots of hidden life.
rock climbing allows me to focus on the problems right in front of me.