A new commercial anti-Japanese encephalitis virus IgM and IgG indirect immunofluorescence test (IIFT) was evaluated for the detection of the humoral immune response after Japanese encephalitis vaccination. The IgM IIFT was compared to two IgM capture ELISAs and the IgG IIFT was analysed in comparison to a plaque reduction neutralization test (PRNT50) and an IgG ELISA. Moreover, the course of the immune reaction after vaccination with an inactivated JEV vaccine was examined. For the present study 300 serum samples from different blood withdrawals from 100 persons vaccinated against Japanese encephalitis were used. For the IgM evaluation, altogether 78 PRNT50 positive samples taken 7 to 56 days after vaccination and 78 PRNT50 negative sera were analyzed with the Euroimmun anti-JEV IgM IIFT, the Panbio Japanese Encephalitis – Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA. For the IgG evaluation, 100 sera taken 56 days after vaccination and 100 corresponding sera taken before vaccination were tested in the PRNT50, the Euroimmun anti-JEV IgG IIFT, and the InBios JE Detect IgG ELISA. The Euroimmun IgM IIFT showed in comparison to the Panbio ELISA a specificity of 95% and a sensitivity of 86%. With respect to the InBios ELISA, the values were 100% and 83.9%, respectively. The analysis of the Euroimmun IgG IIFT performance and the PRNT50 results demonstrated a specificity of 100% and a sensitivity of 93.8%, whereas it was not possible to detect more than 6.6% of the PRNT50 positive sera as positive with the InBios JE Detect IgG ELISA. Thus, the IIFT is a valuable alternative to the established methods in detecting anti-JEV antibodies after vaccination in travellers and it might prove useful for the diagnosis of acutely infected persons. Japanese encephalitis, caused by the Japanese encephalitis virus, is the most prominent viral encephalitis in Asia. Three billion people live in endemic areas and at least 50,000 clinical cases occur each year, although reliable vaccines are available. Concerning the burden caused by this disease, more should be done to prevent it. Good and reliable diagnostics are one of the prerequisites for an effective fight against the virus, but it is nearly impossible to produce and evaluate an in-house assay according to high standard quality criteria as done for commercial tests. Only a few commercial assays are available and the thorough evaluation of these assays is of great importance for diagnostic laboratories. The sensitivity and specificity are statistical measures to assess the performance of a diagnostic assay. In this study the Euroimmun IgM indirect immunofluorescence test (IIFT) was compared to the Panbio and InBios IgM ELISAs. It showed a specificity of 95% and 100%, and a sensitivity of 86% and 93.8%, respectively. The specificity of the IgG IIFT in comparison to PRNT50 was 100% and the sensitivity was 93.8%. Overall, the IIFT showed a comparable performance and could be used as an alternative to the established assays. Flavivirus, family Flaviviridae, is the main cause of viral encephalitis in Asia. Three billion people live in the endemic areas and at least 50,000 clinical Japanese encephalitis (JE) cases occur each year, which are a great burden to the affected populations Japanese encephalitis virus (JEV), a mosquito-borne pathogen of the genus A mouse brain derived and formalin-inactivated JE vaccine (strains Nakayama and Beijing-1), produced by Asian companies, was the predominantly used vaccine for a long time. Only one of these vaccine products (JE-VAX®) was licensed in some non-endemic countries. In recent years Chinese manufacturers have developed a live attenuated and an inactivated vaccine, produced principally on primary hamster kidney cells, and of these the Chinese live attenuated SA14-14-2 vaccine has become widely used in endemic countries Besides the personal protection against mosquito bites, the CDC recommends administration of the JE vaccine to native and expatriate residents of endemic areas, and to travellers staying a month or longer in endemic, especially rural areas during the transmission season The clinical presentation of a JEV infection varies from non-specific febrile illness to severe meningoencephalitis, the main clinical manifestation. Only 1 in 250 to 500 infections is symptomatic. After the incubation period of 4 to 14 days the patients mostly show a rapid onset of fever, headache and gastrointestinal symptoms. Neurological symptoms develop subsequently, with a spectrum ranging from neck stiffness, stupor and impaired consciousness to seizures, parkinsonian movement disorders, convulsions, flaccid paralysis and coma. The mortality rate varies from 8 to 30%, and 30 to 50% of the survivors retain significant long-term neuropsychiatric sequelae The clinical picture of JE resembles the picture of acute encephalitis syndromes of other aetiologies and cannot be differentiated accurately For JE diagnostics “in-house” assays and commercial IgM capture ELISAs are used most commonly. Recently, a commercial indirect immunofluorescence test (IIFT) has become available. The method provides the opportunity to combine different substrates in form of an IIFT mosaic or profile in order to test for other pathogens relevant for differential diagnosis as well as to distinguish cross-reactive antibodies.The aim of this study was to compare the performance of this new commercially available IIFT with other established commercial ELISAs and the “gold standard” in flavivirus diagnostics, the plaque reduction neutralization test (PRNT) in detecting the humoral immune response after immunisation with JE inactivated vaccine. Moreover, the course of specific antibodies after JE vaccination was determined using this new assay.For this present retrospective study a serum collection was examined which was derived from a randomized controlled vaccination study performed by Intercell Biomedical Ltd., Livingston, UK. All details regarding the design of the vaccination study have been published previously The samples from V5 (n = 100) were analyzed for IgG with the Euroimmun anti-JEV IgG IIFT and with the InBios JE Detect IgG ELISA. For IgM altogether 78 samples from V2, V3, V4 and V5 were analyzed with the Euroimmun anti-JEV IgM IIFT, with the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and with the InBios JE Detect IgM capture ELISA.As negative controls for the anti-JEV IgM and IgG IIFT the corresponding samples from V0 were incubated . For IgM an additional 29 samples that had been tested negative in PRNT50 were used as negative controls.Blood donors from northern Germany were used to determine the JEV antibody prevalence in the population detectable with the Euroimmun anti-JEV IIFT. Two hundred IgM samples and 197 IgG samples from blood donors were assessed.A panel of 20 JEV IgG IIFT positive sera from V5 and 10 JEV IgM IIFT positive sera from V3 and V4 was tested with the Flavivirus Profile 2, which allowed the cross-reactivity of anti-JEV antibodies with other flavivirus IIFT substrates to be assessed.In order to determine the possibility of excluding cross-reactivity of anti-dengue positive sera in the JEV assays, a panel of 15 dengue virus (DENV) IgM positive sera was tested with the Euroimmun Flavivirus Profile 2 and a panel of ten dengue virus (DENV) IgM positive sera was tested in the Panbio and Inbios IgM ELISA. Additionally 20 DENV IgG positive sera were assessed in the Flavivirus Profile 2 and InBios JE Detect IgG ELISA.The course of the IgM and IgG response after JE vaccination was analysed in all vaccinees, therefore all PRNT50 positive and selected PRNT50 negative samples were assessed in IgM and IgG IIFT. Six representative courses were chosen and used to present the data in this study.This study was conducted according to the principles expressed in the Declaration of Helsinki. The study was approved by the Ethics Committee of the Medical Association of Hamburg, the Research Ethics Committee of Northern Ireland and the Ethics Committee of the Charité, Berlin. Written informed consent was obtained from all participants and all samples were analyzed anonymously.et al. The PRNT assay was carried out in a 24 well format. Briefly, serial 4-fold dilutions of heat-inactivated serum were incubated in a reaction volume of 1.2 mL for 1 h at 35°C with the JEV strain SA14-14-2. 250 µl of these virus dilutions were plated in triplicate onto monolayers of Vero cells, leading to 40–80 plaque forming units/well. A methylcellulose overlay was used to restrict virus spread. After 5 days of incubation at 35°C, the viral plaques were fixed, stained with crystal violet, and automatically counted . PRNT50 titres (the serum dilution giving a 50% plaque reduction compared to plaque formation in virus-only controls) were calculated using a linear regression (probit) analysis program. PRNT50 titres of≥1∶10 were rated as positive.The PRNT50 was performed by the Intercell AG using a modification of the method described by Sukhavachana The commercial IIFT was produced at EUROIMMUN Medizinische Labordiagnostika AG, Lübeck, Germany, using Vero E6 cells infected with a strain originating form the JEV Nakayama strain (Genebank accession no. EF571853). The infected cells were fixed with acetone before inactivation of the virus by gamma irradiation. In contrast to conventional production methods the cells were grown initially on thin glass slides, which were cut into millimetre-sized fragments (biochips). The biochips were glued onto the reaction fields of microscope slides, offering the possibility to supplement the reaction fields with further biochip substrates. The JEV IIFT assembles one biochip of infected and one of non-infected Vero E6 cells in each reaction field.For evaluation of the anti-JEV IIFT we examined IgM and IgG antibodies in the serum panels using the Titerplane technique as described in the instruction manual. For IgM detection, IgG and rheumatic factors were pre-absorbed with the Eurosorb reagent . Samples were diluted 1∶10, 1∶100 and 1∶1000. 25 µL of the dilutions were applied to the reaction fields of a reagent tray. The slides were then placed upside down into the recesses of the reagent tray, allowing all biochips to come into contact with the drops and the reactions to commence simultaneously under identical conditions without the need of a humid chamber.The cells were incubated for 30 min at room temperature with the serum dilutions. After a washing step with PBS/Tween buffer, either fluorescein isothiocyanat-labeled anti-human-IgG or -IgM was added. Finally, the results were evaluated by fluorescence microscopy with a 200-fold magnification using a 450–490 nm excitation filter and a 515 nm blocking filter, without prior knowledge of the precharacterization of the sera in PRNT50.According to the Euroimmun standard procedure, the fluorescence intensity was rated in levels from 0 (no fluorescence) to 5 (very strong fluorescence) and titres were determined as follows: In the 1∶10 dilution, for example, level 1 was rated as 1∶10, level 2 as 1∶32, level 3 as 1∶100 and level 4 and 5 were rated as more than 1∶100. Titres of ≥1∶10 were considered positive.The Flavivirus Profile 2  was produced using the same method as described for the anti-JEV IIFT. The test combines eight different flavivirus substrates. Five reaction fields in the upper row of a 10-field slide were equipped with four different biochips each, namely with tick-borne encephalitis, West Nile, Japanese encephalitis and yellow fever virus infected cells. Each of the five reaction fields in the bottom row was supplemented with four different biochips containing cells infected with one of the four dengue virus serotypes. Each serum dilution was incubated on these two different reaction fields and evaluated as described above for the anti-JEV IIFT. The assay can be used to determine the predominant flavivirus antibody response and presumptive infection.The Panbio Japanese Encephalitis - Dengue IgM Combo ELISA  is based on the IgM capture format with insect cell expressed and immunopurified JEV and dengue 1–4 antigens. The assay was performed as advised by the manufacturer. 100 µL of the 1∶100 diluted samples were incubated for 1 h at 37°C and after six washing steps 100 µL horseradish peroxidase conjugated monoclonal antibody/antigen complexes were incubated for 1 h at 37°C. The reaction was detected by TMB and read at 450 nm. Panbio Units were calculated as described in the manual and a JE/dengue ratio of the Panbio Units was determined. According to the instruction Panbio Unit >11 are positive and<9 are negative. Panbio Units between 9 and 11 were rated as equivocal and were not considered in the evaluation. This ratio gives presumptive information if the patient is infected either by JEV or dengue virus. A JE/dengue ratio of ≥1 indicates a presumptive infection with JEV and a ratio of <1 indicates a presumptive infection with DENV.The InBios JE Detect IgG ELISA and IgM capture ELISA  were also performed as described in the manual. Both assays rely on the usage of the JE recombinant antigen (JERA), a serological marker which consists of different parts of JEV antigens. All incubation steps were performed for 1 h at 37°C. For the detection step TMB substrate was used, which was measured at 450 nm. For the IgG assay the wells were either precoated with the JERA or with a normal cell antigen (NCA) as a control. 50 µL of the 1∶300 diluted sera were applied to each well.In the IgM-capture ELISA 50 µL of the 1∶100 diluted sera were used in each well. The wells were precoated with anti IgM antibodies. In a second step either JERA or NCA were added and detected with a JERA specific horseradish peroxidase labelled antibody.Immune status ratio (ISR) was calculated by dividing the JERA OD with the NCA OD. IgM ISR values <6 are negative and >10.0 are positive whereas ratios between 6 and 10 are equivocal and were taken out of the evaluation. For the IgG ELISA ISR results >5.0 are rated as positive, values between 2.0 and 5.0 are equivocal and values <2 are negative.The evaluation data of the Panbio Japanese Encephalitis - Dengue IgM Combo ELISA and the InBios JE Detect IgM capture ELISA with sera from infected persons have been published previously All sera were blinded before being tested in IIFT and ELISA and the testing was performed by an experienced technician using the manufacturer's manual. Calculation of 95% confidence intervals (95% CI) was done using the Wilson method For the IgM IIFT evaluation 78 PRNT50 positive samples  were tested in the IgM IIFT, the Panbio IgM ELISA and the InBios IgM ELISA . A negatIn the Panbio IgM ELISA five of the 78 PRNT50 positive samples (6.4%) were rated equivocal and were therefore taken out of the evaluation. The equivocal samples had a geometric mean of the PRNT50 values of 1∶455. 61 of the 73 remaining samples showed concordant results in IIFT and ELISA  whereas 12 samples differed. Five of these 12 samples showed false positive results in comparison to the Panbio ELISA, with low IIFT values from 1∶10 to 1∶32, and seven samples were false negative. The Panbio Units of these seven samples ranged from 13.8 to 24.9.The geometric mean of the PRNT50 values for the 43 positive samples was 1∶535, for the 18 negative samples 1∶109, for the five samples positive in IIFT and negative in ELISA 1∶240 and for the seven samples negative in IIFT and positive in ELISA it was 1∶457.Compared to the Panbio IgM ELISA the IgM IIFT showed altogether a specificity of 95% (95% CI 88.9–97.9%) and a sensitivity of 86% (95% CI 73.8–93.1%) with a positive predictive value of 89.6% (95% CI 77.8–95.5%) and a negative predictive value of 93.2% (95% CI 86.6–96.7%) .In the InBios IgM ELISA eight of the 78 PRNT positive samples (10.3%) could not be diagnosed due to an equivocal result and were not considered in the evaluation. The equivocal samples showed a geometric mean of the PRNT values of 1∶212. Only one sample was equivocal in both IgM ELISAs. 61 samples were concordant  between the ELISA and the IIFT, whereas nine ELISA positive samples were negative in the IIFT with ISR values between 10.6 and 30.1.The geometric mean of the PRNT50 values for the 47 positive samples was 1∶503, for the 14 negative samples 1∶108 and for the nine samples negative in IIFT and positive in ELISA it was 1∶433.Altogether, the evaluation of the IgM IIFT with the InBios IgM ELISA demonstrated a specificity of 100% (95% CI 96–100%), a sensitivity of 83.9% (95% CI 72.2–91.3%), a positive predictive value of 100% (95% CI 92.4–100%) and a negative predictive value of 91.1% (95% CI 83.9–95.2%) .Altogether six samples, positive with both ELISAs did not reveal a positive result in IIFT. Differences in the determination of the specificity were found between both IgM IIFT evaluations. Five samples were negative in the Panbio IgM ELISA, but positive in InBios IgM ELISA and IgM IIFT.The comparison of the results of both ELISAs and the IIFT is demonstrated in the graphical plot . The relAdditionally, the results of the PRNT50 were compared to the results of the IgM IIFT and to both IgM-capture ELISAs. In the Panbio IgM ELISA 64%, in the InBios IgM ELISA 72% and in the IgM IIFT 65% of the 78 PRNT50 positive sera were detected as positive .In the IgG IIFT evaluation, results of the IgG IIFT were compared to PRNT50 values of V5 (56 days post immunisation). 97 of the 100 sera showed a positive PRNT50 result. 91 of these 97 PRNT50 positive sera (93.8%) were detected as IIFT positive with titres of up to 1∶1000 whereas six PRNT50 positive sera were detected as negative by IIFT. The PRNT50 titre range of these six sera was 1∶24 to 1∶182. Two of these sera were IgG and one additionally IgM positive in the IIFT at V4, but declined to, for the IgG IIFT, undetectable levels by V5. All 100 V0 sera and the three PRNT50 negative sera of V5 were detected as negative by the IgG IIFT. In summary, the IgG IIFT showed a specificity of 100% (95% CI 96.4–100%), a sensitivity of 93.8% (95% CI 87.2–97.1%), a positive predictive value of 100% (95% CI 96–100%) and a negative predictive value of 94.5% (95% CI 88.5–97.5%) in comparison to the PRNT50 . Figure Lastly, the results of the InBios IgG ELISA were compared to the results of the PRNT50 at V5. Only 6.6% (n = 6) of the InBios ELISA results were in accordance with the positive results of the PRNT50, whereas 56% (n = 51) of the positive PRNT50 results were not detected by the InBios ELISA. 37.4% (n = 34) of all 91 tested samples were equivocal and a diagnosis was not possible.The geometric mean of the PRNT50 values for the positive samples was 1∶260, for the equivocal samples 1∶421 and for the negative samples it was 1∶198.The amount of cross-reactive antibodies in the anti-JEV positive samples against the different flaviviruses detectable by the Flavivirus Profile 2 IIFT was assessed. Ten JEV IgM IIFT positive sera from V3, V4 and V5 and twenty JEV IgG IIFT positive sera from V5 were tested with the Flavivirus Profile 2. In our study eight of the ten sera showed no cross-reactivity at all, two sera showed cross-reactivity against yellow fever virus, but in both cases the anti-JEV response was dominant. Cross-reactive IgG antibodies were detected in 11 of 20 anti-JEV IgG positive sera. However, again in all sera the anti-JEV response was predominant.Moreover, a panel of 15 dengue IgM and 20 dengue IgG positive samples was tested on the Flavivirus Profile 2 to determine the possibility of differentiation between the cross-reactivity to JEV or other flaviviruses and DENV specific antibodies. On IgM level cross-reactivity against other flaviviruses was observed in four of the 15 sera. Three of these sera showed cross-reactivity against JEV. In all four samples the dengue IgM titre was higher than the titre against the other analyzed flaviviruses (three showed a difference of one and one of three fluorescence intensity stages). Ten of the DENV IgM positive sera were tested with both IgM ELISAs. All sera were diagnosed correctly with the Panbio combined JEV and DENV IgM ELISA as DENV IgM positive. In contrast the InBios IgM ELISA detected five sera as anti-JEV positive, four as equivocal and one sample as anti-JEV negative.A cross-reactive anti-JEV IgG response was observed with the Flavivirus Profile 2 IIFT in all 20 DENV positive sera, but in 80% of the tested sera the anti-DENV IgG response was higher than the anti-JEV response (60% showed a difference of one and 20% of two or more fluorescence intensity stages). All 20 dengue IgG positive sera were tested with the InBios IgG ELISA. 85% of the sera were detected as anti-JEV IgG positive.Furthermore, the anti-JEV antibody prevalence in the northern German population was determined by analyzing a panel of healthy German blood donors with the anti-JEV IIFT. 2% of the sera showed a positive result in the anti-JEV IgM IIFT and 4.1% in the anti-JEV IgG IIFT.The course of the IgG and IgM response after JE vaccination was analyzed additionally. All PRNT50 positive sera (n = 266) were incubated on both IIFTs. Overall 72.6% (n = 193) of these sera were detected as positive by either the IgM or IgG IIFT. IgM IIFT titres were generally lower than IgG titres. On V3 18.2% (n = 8) of the PRNT50 positive sera (n = 44) were IgG positive and 9.1% (n = 4) IgM positive, on V4 the percentage of IgG and IgM positive was 90.8% (n = 89) and 31.6% (n = 31), respectively, and on V5 it was 93.8% (n = 91) and 15.5% (n = 15).94% (n = 94) of the vaccinated individuals (n = 100) developed within the analyzed time span a measurable IgG response, whereas only 33% (n = 33) developed an IgM response detectable with the anti-JEV IgM IIFT. Within the group of the IgM positive sera 94% (n = 31) showed detectable IgM levels at V4, whereas on V5 only 45.5% (n = 15) of the individuals were still positive.Six vaccinees had undetectable IgG titres at all points in time. Three of these six vaccinees were PRNT50 positive for at least two points in time, two were slightly PRNT50 positive (up to 1∶54) for at least one point of time and one was PRNT50 negative. All six vaccinees showed likewise negative IgM IIFT results.In 84.8% (n = 28) of the vaccinees with an IgM response (n = 33) IgM and IgG titres were detectable simultaneously. Only in 9.1% (n = 3) of these vaccinees the IgM reaction was detectable before the IgG response. The PRNT50 titre range of the sera undetectable in IgM IIFT was 1∶10 to 1∶1965 , and for the sera undetectable in IgG IIFT it was 1∶10 to 1∶317 .In In 36.4% (n = 12) of all IgM positive vaccinees the IgM and IgG titres descended simultaneously, as in vaccinee no. 3. In this case the PRNT50 titre displayed the same course as the IgM and IgG titre. A simultaneous course of all measurable titres was also seen in 40% (n = 40) of the analyzed vaccinees, in 26% (n = 26) of these the PRNT50, IgM and/or IgG titres descended simultaneously and in 14% (n = 14) all measurable titres ascended simultaneously over time. Generally, in 66% (n = 66) of the vaccinees the PRNT50 titre declined towards the end of the study. In vaccinees no. 4 and 5, as in 30.3% (n = 10) of all IgM positive vaccinees, a decline of the IgM titre was observed while the IgG titre was constant until the last analysis. In 12.1% (n = 4) of all IgM positive vaccinees, as also seen in vaccinee no. 6, the IgM titre declined while the IgG titre increased over the analyzed time span.The performance of the new anti-JEV IIFT was compared to two different IgM-capture ELISAs and the PRNT50.Overall, the anti-JEV IgM IIFT showed a performance comparable to the IgM-capture ELISAs. The difference in specificity of the IIFT found in comparison to the Panbio and the concordant results compared to the InBios assay might be due to a difference in the sensitivity of the respective ELISAs. The Panbio ELISA revealed in the published evaluation study In direct comparison both IgM capture ELISAs were more sensitive than the IgM IIFT. This phenomenon has been detected in several IIFT evaluation studies and might be explained by different sample amounts, different antigens used for the assays and differences in the presentation of the antigen. Both ELISAs relied on recombinant antigens, whereas in the IIFT complete virus was used. However, similar numbers of PRNT50 positive samples have been detected as positive by all three methods. Thus, the IgM IIFT represents another diagnostic method which is rapid to perform and especially suitable for small serum numbers. An additional advantage of the IIFT is the possibility to identify and exclude unspecific reactive sera by recognizing the unspecific staining of the non-infected control cells.The anti-JEV IgG IIFT revealed 56 days after vaccination a sensitivity comparable to the PRNT50, but it is much faster and easier to perform than the time-consuming PRNT50 as the performance of the IIFT takes only 1.5 hours. In contrast, the InBios IgG ELISA revealed in this study an inadequate detection of PRNT50 positive samples and is therefore not suitable to determine the antibody response after JEV vaccination.Although the actual protective immunity can only be determined by the neutralization assay, the IgG IIFT offers a possible alternative to assess the anti-JEV IgG titre after vaccination. This might for example be a valuable tool for a rapid diagnosis of the immune status in travellers. Nevertheless, the limitation of the use of the IIFT in assessing the antibody response after JE vaccination lies in the presence of flavivirus cross-reactive antibodies from previous infections. Therefore, the intensity of the detection of cross-reactive antibodies in different serum panels was studied.The Flavivirus Profile 2 was used to determine the level of cross-reactivity detected in IIFT in the JE-vaccinated persons. In eight of the ten sera no cross-reactivity at the IgM level was found. Similar results have been described in the literature The results of the DENV positive panel tested in all three assays revealed that even the use of a recombinant antigen as found in the Panbio and InBios ELISA cannot prevent cross-reactivity. The high cross-reactivity shown by the Inbios IgM ELISA was in accordance with the results published previously Nevertheless, to differentiate between a specific and a cross-reactive immune response in the case of an acute infection a fourfold increase in the titre of a consecutive serum should be demonstrated. However, a single IgM positive result can also be confirmed by other techniques such as PRNT. Additionally, NS1 antigen detection could be a valuable tool for the early diagnosis of Japanese encephalitis virus infections. In dengue infection e.g. NS1 is detectable very early, before IgM is produced and up to 9 days after the beginning of symptoms. The overall diagnostic sensitivity in early stages of the dengue disease was increased by the combination of NS1 and IgM detection The detection of anti-JEV IgM and IgG antibodies in the northern German population could be explained by cross-reactive antibodies against other flaviviruses, for example due to vaccination against tick-borne encephalitis or yellow fever. Therefore, also in areas without JEV and DENV occurrence it should be taken into account that cross-reactive antibodies against other flaviviruses can alter the serological results. Additionally, in patients with a relevant travel history the occurrence of dengue antibodies has to be considered.Furthermore, the course of specific antibodies after JE vaccination detectable by IIFT was determined. Altogether only 33% of the vaccinated persons established an IgM response detectable in IIFT. Therefore, if the detection of the anti-JEV titre after vaccination is needed, it might be advisable to test for IgG rather than for IgM. Since on day 28 after vaccination the IgG response was at 18% still rather low, the best time to determine the IgG titre was from 35 days after vaccination onwards.It is expected that in sera from acutely infected persons the fraction of sera detected as IgM and also IgG positive in the IIFT will be higher, as their serological immune response is stronger. But this has to be evaluated with a panel of sera from persons from endemic areas infected naturally with JEV.Overall the new Euroimmun JEV IIFT showed comparable results to the commonly used commercial IgM capture ELISAs and the “gold standard” in flavivirus diagnosis, the PRNT50. It is a valuable tool for analyzing the immune response after vaccination in travellers and people resident in endemic areas and it might prove useful for the diagnosis of acutely infected persons.Checklist S1STARD Checklist(0.05 MB DOC)Click here for additional data file.Flowchart S1STARD Flowchart(0.03 MB PDF)Click here for additional data file.


Individuals have increasingly sought complementary therapies to enhance health and well-being during cancer, although little evidence of their effect is available. how an Iyengar yoga program affects the self-identified worst symptom in a group of participants. whether quality of life, spiritual well-being, and mood disturbance change over the Iyengar yoga program and at 6 weeks after the program. how, from a participant’s perspective, the Iyengar yoga program complements conventional cancer treatment.We investigatedThis pre–post instrumental collective case study used a mixed methods design and was conducted at a private Iyengar yoga studio. The sample consisted of 24 volunteers  who were currently on treatment or who had been treated for cancer within the previous 6 months, and who participated in ten 90-minute weekly Iyengar yoga classes.The main outcome measures were most-bothersome symptom , quality of life and spiritual well-being , and mood disturbance (Profile of Mood States–Short Form). Participant perspectives were obtained in qualitative interviews.t(23) = 5.242; p < 0.001), quality of life  = 14.5; p < 0.001), spiritual well-being  = 14.4; p < 0.001), and mood disturbance  = 10.8; p < 0.001) during the program. At follow-up, quality of life (t(21) = −3.7; p = 0.001) and mood disturbance (t(21) = 2.4; p = 0.025) significantly improved over time. Categorical aggregation of the interview data showed that participants felt the program provided them with various benefits not included on the outcomes questionnaires.Statistically significant improvements were reported in most-bothersome symptom (Over the course of the Iyengar Yoga for Cancer program, participants reported an improvement in overall well-being. The program was also found to present participants with a holistic approach to care and to provide tools to effectively manage the demands of living with cancer and its treatment.


In light of the child overweight and obesity problem in Australia, resistance training programs have been trialled as an innovative way of assisting children increase lean body mass and reduce body fat. The purpose of this study was to investigate the factors influencing overweight children's participation in a resistance training trial program.Parent-child pairs who participated in the trial program were invited to take part in a follow-up individual interview to discuss their program experiences. In total, 22 semi-structured interviews were conducted with 11 parent-child pairs.The factors found to be most relevant to program commencement among parents were a desire for their child to lose weight and gain confidence, the proximity of the venue, and no cost for participation. For children, the most relevant factors were the opportunity to build strength and improve fitness and having supportive parents who facilitated program initiation. The factors most relevant to continuation for parents were the quality of the program management, being able to stay for the sessions, the child's improved weight status, coordination, and confidence, and no cost for participation. Weight loss and improved confidence were also motivators for continuation among the children, along with pleasant social interaction with peers and trainers and ongoing parental support.Different factors variably influence program commencement and program continuation in both parents and children. This has important implications for future interventions that aim to successfully recruit and retain intervention participants. Overweight and obesity in Australia constitute a widespread health problem that has been named a national priority by the National Preventative Health Taskforce . Child oOverweight and obese children can experience numerous problems as a result of their weight status. These include neurological , gastroeExercise training, which encompasses both resistance and aerobic exercise, has been found to be beneficial in promoting healthy body composition and weight control for children aged six to 18 years ,14. WhilResistance training programs can also have positive psychological outcomes for children. In particular, they have been shown to have a positive effect on physical self-perception in young people ,23. An eDespite the benefits of physical activity, it can be difficult to encourage exercise initiation among overweight and obese children . While pGiven the relative recency of resistance training programs for children and our poor understanding of the factors that influence participation in such programs, the purpose of the present study was to explore the barriers, motivators, and facilitators to overweight and obese children commencing and continuing a trial resistance training intervention program conducted in Perth, Western Australia. As children and their parents are likely to influence program participation , both grThe children involved in the study participated in a resistance training program designed for overweight and obese children aged 6 to 13 years. Ethics clearance for the study was obtained from the Edith Cowan University Human Ethics Research Committee . Participants were randomly assigned to one of three groups that varied in program duration: 8 weeks, 16 weeks, and 24 weeks. The physiological results of the study can be found elsewhere , but in Participants were recruited into the program over a period of two months through local general practitioners and an advertisement in the local community newspaper. Sixty-three children were screened for inclusion, at which stage 13 were deemed ineligible. The resistance training component of the trial program was conducted by third-year undergraduate exercise science students at the University under the supervision of the chief investigators. Parents could either stay to observe their children during sessions or return to collect them later. In total, 31 families took up the opportunity to participate in the program. Six (19%) of the children completed the 8 week program, nine (29%) completed the 16 week program, and 16 (52%) completed the 24 week program. On average, 87% of sessions were attended by participants. The attrition rate by the end of the program was 87%, with only four of the 31 children who commenced the program enrolling in the subsequent fee-paying version.Eleven parent-child pairs participated in semi-structured individual interviews after completing the trial program. All families that started the program were invited to participate, including those who finished the program and those who withdrew. Of the participating children, one had completed the 8 week program, two had completed the 16 week program, and eight had completed the 24 week program. Table Parents and children were interviewed on the same day but separately to avoid contamination of responses. Informed consent was obtained from both the parents and the children. The interviewers had not previously met the interviewees and were not involved in program delivery, which encouraged the interviewees to give expansive and descriptive accounts of their experiences with the program. During the interviews, a wide range of topics was discussed including motivations to commence the program, perceptions of the program, and any problems that may have been experienced. Interviewees were also asked to reflect upon the positive and negative aspects of the program and to describe any barriers to their ongoing participation in similar programs in the future.The interviews were digitally recorded with the interviewees' informed consent. The recordings were transcribed verbatim and imported into NVivo8  for coding and analysis. Content codes were created to cover the topics listed in the interview guide and emergent concepts that were identified during analysis. Demographic characteristics of the interviewees were also captured.All the adult interviewees and all but one of the child interviewees reported highly favourable experiences with the program. Only one child reported not enjoying the program, and this was attributed by both himself and his mother to his general dislike of exercise and strong preference to be sedentary. Both the parents and the children were able to discuss at length the factors that contributed to their decisions to commence and continue with the program. These factors are summarised in Table When discussing the factors that led them to commence the program, the interviewees focused on anticipated outcomes and the proximity of the training venue in relation to the participants' homes. These two themes are outlined below.The parents reported that their primary motivation for enrolling their children in the resistance training program was for their children's health to improve through weight loss This was in accordance with the benefits stated in the advertisement for the program featured in the local newspaper, which included specific mention of the goal of reduced body fat. It was therefore not surprising that this factor emerged as the dominant motivator:Sean's mother, 24 weeks)I think we could see that Sean was getting just a little bit of puppy fat and we thought we could strengthen him up. And also, he is a big boy so it would be nice to get his muscles moving a bit. I think it was that she wasn't doing very much in the way of exercise. She's a fairly solid sort of build, that sort of concerns her mother a little bit - partly because her father is a very solid build, quite overweight. So her mother is sort of worried about Sarah a bit, and so that was the motivation initially (that) came from her mother. And Sarah was happy to go along to it. I doubt very much that it was expressed in those terms to Sarah. Probably more the weight loss. Like I'd read they'd sort of said, you know, we're looking at children who are overweight and Tiffany was quite overweight for her height at the time. So it was like, "Oh great, this sounds like a program specifically tailored for Tiffany". What were your expectations of the program? Basically for Larry to get healthy and to lose the extra weight he'd put on. I think a lot of people were looking at improving self-esteem. Yeah, yeah it was good from that point of view, of self-confidence and self-esteem. Well, I always wanted to get a bit faster because usually in the sports carnivals I always come last, so I wanted to become a bit faster. A bit of strength because my Dad always tried to push me down, but he always won and I wanted to beat him one time at least. My parents, I think they said, "Sean, would you like to do this program?"...They said, "You can go in and do some weights to build up some muscle", and I thought, "Oh yeah, I'll do that". It sounded interesting, and for health and fitness reasons. Just because I wanted to lose weight and get healthier. ...I think it was more my parents wanted me to do it. My mum was the one who told me about it and I think there was an ad somewhere and she said, "Why don't you decide to do this? It will be good for you." For those families living close by, a facilitator to enrol in the trial program was the close proximity of the facility to their place of residence. Most of the families lived near the University, (i.e. within five kilometres), and only few lived further afield (i.e. up to 25 kilometres). Proximity was perceived to be important because of the competing schedules of different family members that needed to be accommodated to allow attendance:Elizabeth's Mother, 24 weeks)We were living only five minutes away, so we could drop her and then come back if we had things on. And some days a friend might drop her off if I was coming late home from work. So yeah, it worked in really well... We were lucky we were just living in (nearby suburb), so it was really good. (The man) who was running it, he was great. What he did was fantastic...he deserves a medal that guy. (The program manager) understood that only a few of us could come in the morning, so like when Tiffany had to do a re-scan he would make sure that whether it was him or someone else, that someone was there to do the scan in the morning. So that worked really well. He would come at 4 pm and they wanted to change it to 4:30 pm. I said to them, "Look, if I go home, if he finishes school, goes home, gets changed, sits on the couch, eats something, then he's not going to want to go anywhere." And the guy that was actually training him said, "Oh it's all right, just bring him in at 4 pm and we'll just train him at 4 pm." So that was another reason for me to go, because I knew I would be catching up with (friends), whereas I wouldn't normally see them... Sometimes when I said, "Oh, I really don't want to go this week", and I knew I should because it was a research program for the students here. And I did enjoy it a lot, and I knew it would be fun, and I'd always end up having a good time. I thought it was really good. I mean like, at the beginning I thought I was just going to do it to lose a bit of weight, but in the end I really enjoyed it because I made friends and yeah, I also lost weight. No, he didn't make friends with any of the kids. He more made friends with the students who were running the program. And then we used to see them at the shops and things like that and say, "Hello", so that was good...They used to have a laugh, and a joke, and they liked that. The trainers were excellent with the kids. They were really, really kid orientated. So they made it lots of fun while they were doing all the work. Researcher: What were your favourite parts of the program? I suppose the actual students that were running it, they were really friendly. As you'd wait there to start the program they'd always have a big smile, "Hello, come on in and we'll start up." ...They helped us enjoy it and they were really good. I tended to sit in the room and watch so I could try and see how they were telling her how to do the exercises properly, and the stretches. Mum blackmailed me with things like computer games and a computer and stuff like that. I actually enjoyed it, I thought it was good. I enjoyed the exercises and knowing what I can and can't do, and being able to...I enjoyed challenging myself and being able to know more about myself and what I am capable of, and yeah, it was really good. She did lose quite a bit of weight at the time. .It was pretty good. It actually helped Liam to lose weight. He did really well, like with the DEXA scans and all that sort of stuff, and like you know, he'd lost five percent body fat in the research program. If you're really like obese, like I was, you lose heaps of weight and you just feel good. I think confidence was a really big thing. She got more enthusiastic as she was doing the exercises and things like that. I think that confidence is very important to children that may be a little bit chubbier than others, you know. And it's important that they get more comfortable with their physical self. Once you start feeling better and stronger, well then you go on to more things. He doesn't like to do team sport. He's not that good at team sports or anything like that. It's really repetitive and I just know that whenever we tried to get him to do something he wouldn't. He was like, "Oh no, I might not be good enough", and all this sort of stuff. So the reason for this was just to get him to do something, but it was an individual thing as well. So it was just him and he knew he could do it. Like once he started doing it, he knew he could do it and he didn't have to please anybody else. It was all about himself. I enjoyed challenging myself and being able to know more about myself and what I am capable of. ...you just feel good about yourself; you don't feel like different to others. She's not tremendously well coordinated and her coordination improved, and her overall confidence. Her coordination has gotten better and her balance in general. I would have loved to have kept doing the program, and I would probably be doing it now, I just can't afford to do it. Yeah, she really enjoyed it, she was hoping there was going to be another one, but they said they had to pay for the next one. So yeah, we just couldn't afford it. I came along to all of the sessions that they had. I would have liked to have continued it, but I think after the initial training sessions they did offer it, but it had a fee. So, and that's sort of what's holding us back at the moment, because I can't afford it at this point in time. It was all good except the only thing was there was nowhere for the kids to go and exercise in a similar program after it finished. I'm on a single income so I cannot afford to pay for Larry to do another program like this one.  items to address child obesity would seem imperative if the Australian Government is to address what is an increasingly expensive social and financial problem.The study findings demonstrate that different factors variably influence program commencement and program continuation in parents and children. This has important implications for future interventions that aim to successfully recruit and retain intervention participants.Given the qualitative nature of this study, it is not possible to determine the extent to which the findings are representative of the views of all the program participants and their parents. In particular, there is little representation of families from the 8 and 16 week programs. Also, the study did not explore the barriers for commencement that prevented other families from enrolling in the trial program in the first instance. Given the repetitive nature of resistance training programs, it is a further limitation of the study that the issue of boredom was not addressed with the interviewees. However, the interview data provide insight into numerous factors that can influence program commencement and continuation, providing program designers and managers with important information to consider in the development of future recruitment and retention strategies for resistance training programs.The authors declare that they have no competing interests.MRM, RUN, and SP participated in the design of the study. MP and SP conducted the interviews and carried out the thematic analysis of the transcripts. All authors assisted in drafting the manuscript and read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1471-2458/10/709/prepub


Ergosterol has been considered the “fungal sterol” for almost 125 years; however, additional sterol data superimposed on a recent molecular phylogeny of kingdom Fungi reveals a different and more complex situation.5 sterols in the earliest diverging fungal species to ergosterol in later diverging fungi. There are, however, deviations from this pattern in certain clades. Sterols of the diverse zoosporic and zygosporic forms exhibit structural diversity with cholesterol and 24-ethyl -Δ5 sterols in zoosporic taxa, and 24-methyl sterols in zygosporic fungi. For example, each of the three monophyletic lineages of zygosporic fungi has distinctive major sterols, ergosterol in Mucorales, 22-dihydroergosterol in Dimargaritales, Harpellales, and Kickxellales (DHK clade), and 24-methyl cholesterol in Entomophthorales. Other departures from ergosterol as the dominant sterol include: 24-ethyl cholesterol in Glomeromycota, 24-ethyl cholest-7-enol and 24-ethyl-cholesta-7,24(28)-dienol in rust fungi, brassicasterol in Taphrinales and hypogeous pezizalean species, and cholesterol in Pneumocystis.The interpretation of sterol distribution data in a modern phylogenetic context indicates that there is a clear trend from cholesterol and other Δhttp://aftol.umn.edu.Five dominant end products of sterol biosynthesis , and intermediates in the formation of 24-ethyl cholesterol, are major sterols in 175 species of Fungi. Although most fungi in the most speciose clades have ergosterol as a major sterol, sterols are more varied than currently understood, and their distribution supports certain clades of Fungi in current fungal phylogenies. In addition to the intellectual importance of understanding evolution of sterol synthesis in fungi, there is practical importance because certain antifungal drugs  target reactions in the synthesis of ergosterol. These findings also invalidate use of ergosterol as an indicator of biomass of certain fungal taxa . Data from this study are available from the Assembling the Fungal Tree of Life (AFTOL) Structural and Biochemical Database:  The analyses support a monophyletic group of organisms with more diversity in the early diverging lineages than previously recognized 28 sterols common in most fungi.As in cholesterol biosynthesis in animals, lanosterol  and 3, i28 sterol ergosterol vary according to the sequence of double bond transformations. In some taxa, the pathways to ergosterol are incomplete and in some cases result in the formation of other end-products . A second methylation resulting in a 24-ethylidene, which is reduced to 24-ethyl, leads to C29 sterols in some taxa. The pathways for the five major sterol end-products, and C29 sterols, are summarized in Multiple pathways leading to the formation of the CThe distribution of sterols in the kingdom Fungi from the early diverging lineages, including zoosporic and zygosporic forms, to the most advanced taxa of the Ascomycota and Basidiomycota is discussed in light of recent phylogenetic analyses Sterols in fungi typically exist as a mixture of several sterols with one that is dominant, i.e., representing over 50% of the total sterol composition. The dominant or major sterol is most often accompanied by other sterols that are typically intermediates in the synthesis of the major sterol. The discussion of the distribution of sterols herein is in the context of the major or dominant sterol that accumulates in a fungus . For cla1NMR) are given the most weight as opposed to less specific methods .A variety of methods were used in the isolation and characterization of sterols discussed in this paper as applied by the authors of the literature cited herein. Where anomalies exist, identifications based on the most advanced instrumentation  Structural and Biochemical Database <Rozella and Olpidium), occur outside of these phyla in independent lineages with the implication that flagella were lost on several occasions The zoosporic fungi (chytrids) are now placed among three phyla . Other flagellated species, both endobiotic parasites  is not well-resolved, and they have been maintained as separate subphyla rather than united in a single phylum This lineage of zygosporic fungi  appears to be a monophyletic group Ergosterol is absent, however, from Mortierellales. The distribution of major sterols varies among Mortierellales depending on the species, but includes desmosterol, 24-methylene-cholesterol, and 24, 25-methylene-cholesterol or combinations of these sterols Dimargaritales, Harpellales, and Kickxellales (DHK Clade). The DHK clade of the zygosporic fungi includes Dimargaritales, Harpelles, and Kickxellales  have been investigated, and both are reported to have ergosterol as their major sterol Z. insidians was reported to be a pythiaceous oomycete, and the presence of ergosterol supports the placement of this species as a fungus rather than an oomycete. The Oomycetes in which pythiacious fungus-like organisms are placed do not produce sterols The Zoopagomycotina may also belong in the DHK clade (see below), but we have maintained it in a separate lineage for now Conidiobolus and Entomophthora species analyzed contained 24-methyl cholesterol as the major sterol  has been reported as the major sterol of four species of the phylum, and ergosterol has not been detected The Glomeromycota, arbuscular mycorrhizal fungi, are associated with the roots of many plants. More recently, 24-ethyl cholesterol  as their primary sterol. Schizosaccharomyces pombe produces ergosterol This ascomycete subphylum is varied in many of its morphological and biochemical characters, including the major sterol production. Saccharomyces cerevisiae contain ergosterol as the major sterol Several strains of Tuber and Terfezia have in common with Taphrinales the accumulation of brassicasterol as a major sterol Tuber contains a mixture of ergosterol and brassicasterol , each in a different genus, differ from other Pezizomycotina by their production of 24-methyl-cholesta-5,24(28)-dienol (24-methylene cholesterol) casterol .7 > Δ5,7 > Δ5) to complete the synthesis to the end-product, i.e., Δ5, do not occur in the rust fungi, resulting in Δ7 and Δ7,24(28) sterols.This diverse taxon contains among other organisms a large group of plant parasites, the rust fungi . The rust fungi contain 24-ethyl-cholesta-7,24(28)-dienol -dienol) or 24-ethyl-cholest-7-enol (stigmast-7-enol) as major sterols The smut fungi  contain ergosterol as the dominant sterol . As withCoriolus sanguineus, Fomes applanatus, and Polyporus pargamenus) in the core polyporoid clade Ergosterol is the major sterol in members of the Agaricomycotina, except for three taxa . In the case of the mycorrhizal fungi, dThere is a progressive shift from considerable structural diversity of dominant sterols among fungal taxa to a single dominant sterol, ergosterol. Cholesterol does not appear to be produced by the later diverging fungi, and ergosterol is not found in the earliest diverging lineages . In viewThe formation of ergosterol requires three reactions that are not involved in cholesterol biosynthesis, i.e., 1) methylation at C-24, 2) reduction at C-24(28), and 3) insertion of the double bond in the side-chain at C-22. The ability to alkylate at C-24 appears to have occurred early in fungal evolution since C-24 methyl and ethyl sterols occur in some of the chytrids. It seems plausible that the ability to produce ergosterol evolved through a series of progressive steps corresponding to the biosynthetic steps leading to this sterol, and that the intermediates accumulated as major sterols in evolutionarily intermediate taxa. The dominance of 22-dihydroergosterol and the absence of ergosterol in Kickxellomycotina (DHK clade of zygosporic fungi) supports the hypothesis that intermediates in ergosterol biosynthesis may accumulate in taxa from which ergosterol-producing taxa evolved.5,7 configuration) and C-22 in ergosterol The selection pressures involved in the evolutionary change from cholesterol to ergosterol as a primary component in fungi are not understood. The driving force for the evolution of ergosterol rather than cholesterol is not intuitive for several reasons. More energy is required for ergosterol synthesis than for cholesterol, and in addition cholesterol has been shown to be the most functionally competent structure in artificial membrane systems Multiple functions for sterols have been identified in yeasts This study has helped to clarify the available information on sterol distribution in the kingdom Fungi by placing it in a modern phylogenetic context and has identified areas for additional sampling (both for sterols and genes), especially of certain fungal clades in the early diverging lineages and core polypore taxa. It will be useful to clarify more fully how sterols are distributed among fungi and sister taxa Table S1Species sampled from the literature and their major sterols listed in phylogenetic arrangement.(0.09 MB DOC)Click here for additional data file.


An integrated computer system consisting of an expandableionanalyzer and a PC has been used to automate concentrationmeasurements with ion-selective electrodes (ISEs). The ionanalyzerdetermines ionic concentrations using a reference electrodecoupled with an ISE. The measurements and calibrations arecarried out in a thermostated sample changer equipped with a flowcell. The data obtained by the ionanalyzer are sent via a standardRS 232-C interface to a microcomputer. In this paper, we describethe automatic data acquisition system and the subsequent processingof the measurements. One program (Transorion) automaticallycollects the measurements carried out by the ionanalyzer, giving areal-time graphic representation of the measurement on thecomputer screen. A second program (Graforion) facilitates themanagement of the data stored by the first program, and listing andgraphics of these can be obtained on the printer/plotter. The methodhas been used to study continuous concentration changes in anaqueous solution of potassium iodide.


Implementation of linkage to HIV care programs in the U.S. is poorly described in the literature despite the central role of these programs in delivering clients from HIV testing facilities to clinical care sites. Models demonstrating success in linking clients to HIV care from testing locations that do not have co-located medical care are especially needed.Data from the Antiretroviral Treatment Access Studies-II project ('ARTAS-II') as well as site visit and project director reports were used to describe structural factors and best practices found in successful linkage to care programs. Successful programs were able to identify recently diagnosed HIV-positive persons and ensure that a high percentage of persons attended an initial HIV primary care provider visit within six months of enrolling in the linkage program.Eight categories of best practices are described, supplemented by examples from 5 of 10 ARTAS-II sites. These five sites highlighted in the best practices enrolled a total of 352 HIV+ clients and averaged 85% linked to care after six months. The other five grantees enrolled 274 clients and averaged 72% linked to care after six months. Sites with co-located HIV primary medical care services had higher linkage to care rates than non-co-located sites (87% vs. 73%). Five grantees continued linkage to care activities in some capacity after project funding ended.With the push to expand HIV testing in all U.S. communities, implementation and evaluation of linkage to care programs is needed to maximize the benefits of expanded HIV testing efforts Testing, linkage to care, and HIV treatment have been recognized as the three arms of an approach to HIV prevention receiving recognition and funding recently in the U.S. . HoweverPrograms that actively facilitate connecting a recently diagnosed HIV-positive person to HIV medical care are known as linkage to care programs or models. Linkage to care models have been mostly homegrown, small single-site efforts at large medical centers closely co-located with HIV testing facilities. Some of these models from large medical centers with emergency room testing and a nearby HIV clinic have shown impressive rates of linkage to care within a few months, often exceeding 85% -11. HoweCDC funded a linkage model incorporating multiple sources of clients and testing locations ; the study used a two-arm randomized controlled trial that compared a brief strengths-based case management intervention with standard-of-care referral in linking recently diagnosed HIV-infected persons to a primary medical care provider . ParticiBased on positive outcomes from the ARTAS-I trial, CDC funded a demonstration project (ARTAS-II) to evaluate the feasibility of implementing that same linkage to care model in local and state health departments and non-profit community-based organizations (CBOs). Ten health departments or CBOs were funded to implement the linkage to care program. Results from ARTAS-II showed that 79% of all participants enrolled in the program visited an HIV clinician at least once within the first six months .These findings indicated that a brief linkage case management intervention can be implemented effectively by service-oriented organizations in typical HIV programs in rural, mid-sized, and urban settings in the U.S. The objective of the current report was to describe best practices and structural characteristics of organizations from the ARTAS-II demonstration project, specifically those factors which facilitated successful implementation of a program to link recently diagnosed HIV-infected persons to HIV care providers.The best practices, characteristics, and structural factors in this report incorporate information obtained from the 10 ARTAS-II study sites from October 2004 to June 2007 5. Conduct case management as an active, community-based activity Grantee and implementing site characteristics, enrollment totals, and rates of linkage to HIV care are presented in Table In the application process, grantees were competitively selected from health departments in the southeastern United States and non-profit CBOs throughout the U.S. Of the 10 funded grantees, there were 4 state health departments, 1 county health department, and 5 CBOs.Following the funding award, grantees selected an appropriate agency to implement the linkage program. Based on the grantees' final reports, the agencies best suited to implement such a program were health departments  or CBOs with strong, established working relationships with high-volume HIV testing sites.For the four state health department grantees, a staff person located in the office of the state AIDS director typically served as the project director. This staffing decision ensured that the project director was familiar with the statewide Ryan White HIV/AIDS Treatment Modernization Act programs and was in a position to select the best agency to implement the linkage program. None of the state health department grantees directly implemented the linkage program; instead, state health departments selected CBOs or local health departments to serve as the implementing organizations. Project directors selected implementing organizations through a non-competitive process, which resulted in the selection of capable and experienced service agencies to implement the linkage program. Collectively, the four state health department grantees enrolled an average of 74 participants compared with non-state health department grantees that enrolled an average of 55 participants . Other factors considered when selecting an implementing organization were the organization's level of interest and commitment to offering linkage to care services to HIV-infected clients and their capacity to collect and manage data that would be used to evaluate the linkage program. Lastly, the project directors at state health departments considered selecting organizations in regions of the state that had the highest or increasing rates of new HIV infections. In Louisiana, for example, HIV/AIDS program staff reviewed counseling and testing data to select a region of the state in which to implement the linkage to care program. Once the Baton Rouge region was selected, these data were used to identify potential referring sites  that tested a high volume of clients and had high positivity rates.Other sites considered the ease with which the linkage to care program could be integrated into existing HIV care delivery systems. In South Carolina, for example, the Department of Health chose to implement the linkage program in two regions of the state where there was only one Ryan White Part B provider, which streamlined integration of the linkage program with HIV prevention and care services in those regions.Five grantees were non-profit CBOs, and in all cases that same CBO served as the implementing agency. Two directly-funded CBO grantees that we highlight in this report are the Kansas City Free Health Clinic and Health Services Center (Alabama). Both these CBOs offered co-located comprehensive HIV/AIDS services, as outlined above. In addition, these two CBOs had pre-existing, strong working relationships with the local health departments serving their community, which facilitated the process of securing cooperation from health department disease intervention specialists (DIS) and counseling and testing staff to supply referrals of recently diagnosed HIV-positive persons. One common tactic used by CBO staff to market the linkage to care program to health departments was to emphasize the reduced burden on health department staff for linkage to care, thus freeing up staff to perform other core job duties, such as partner services.One of the logistical challenges encountered by CBOs was the lack of office space at local health departments for housing the linkage coordinators (persons delivering the linkage intervention to clients). The CBO in Alabama, for example, addressed this issue by securing an agreement with the local health department to pay monthly rental assistance for onsite office space within the health department to house the linkage coordinators. This onsite office space allowed the linkage coordinators to be nearby to respond quickly to referrals and meet with clients soon after receiving an HIV-positive diagnosis at the health department. In sum, pre-existing relationships with local public health authorities provided a foundation for the goodwill needed for CBOs to secure consistent referrals of recently diagnosed HIV-infected persons.One key step in initiating a linkage to care program was for the implementing agency to identify appropriate agencies and organizations in the community with which to partner. These collaborations involved partnering with 1) agencies conducting HIV testing, 2) HIV primary care providers to whom clients could be linked to medical care, and 3) case management agencies to which clients could be referred for long-term case management services after completing the brief linkage to care intervention. One structural attribute that facilitated successful implementation was having as many HIV services as possible  co-located within the same building or campus where the linkage case management intervention was being delivered. Sites that had co-located services averaged 87% of participants linked to care, whereas sites without co-located services averaged 73% of participants linked to care  enrolled 1 to 2 persons per month into the linkage program. To counteract this period of slow enrollment, the Virginia Department of Health chose to mandate that all DIS staff in the central Virginia region refer all persons testing HIV-positive to the linkage to care program. After the DIS referral mandate was implemented, the CBO averaged over 6 enrollments per month during the next 4 months. The state health department actively monitored DIS referrals to the linkage program and created a mechanism for receiving feedback from the linkage program (a letter mailed back to the DIS) to indicate whether or not the client had been successfully linked to HIV primary care.In South Carolina, the implementing agency in Greenville had an HIV prevention services employee who essentially functioned as part of the DIS team and had office space in the local health department, so the referral process was relatively seamless. However, due to the large size and complex structure of the health department in Columbia, planning and coordination was required to ensure that DIS and counseling and testing staff were actively referring persons testing HIV-positive to the linkage program. For the first several months of the project, the linkage coordinators and their supervisors met frequently with health department staff to remind them of the new protocol for contacting the linkage coordinators when HIV-positive test results were to be delivered. Once the linkage coordinators established a presence at the clinic, health department staff began consistently referring patients to them.In Louisiana and Kansas City, linkage program staff initiated contact with all of the counseling and testing sites in the area, met with key staff, presented program information at staff meetings, and distributed brochures, referral forms, referral flow charts, and other relevant information regarding the linkage program. Many of the counseling and testing sites in Baton Rouge were already familiar with the two CBOs implementing the linkage to care program because these CBOs provided long-term HIV/AIDS case management services. The trust and familiarity with these two CBOs increased cooperation between local counseling and testing sites and linkage program staff in Baton Rouge.In Alabama, Kansas City, and Virginia, there was some initial resistance among DIS staff at the health departments about relinquishing control of the referral to care process. To alleviate DIS concerns about overlapping job duties, the linkage coordinators emphasized that their role was focused on linking clients to HIV medical care, which would relieve DIS of this additional task and allow them to focus on core job duties such as partner elicitation. DIS staff were generally cooperative if they viewed the linkage coordinator's role as complementary and supportive rather than duplicative of their role. Over time, as the linkage coordinators demonstrated their commitment and competency in providing linkage to care services, DIS staff began trusting them with all referrals of recently diagnosed HIV-positive persons.Local hospitals conducting HIV testing in the emergency department or inpatient units were not a major source of referrals during the CDC demonstration project, but these venues can provide a steady source of referrals to linkage programs. Given CDC's revised recommendations for HIV testing and the expansion of rapid testing, emergency departments will likely become a more important source of HIV-positive diagnoses . Since twithout co-located HIV primary medical care and ARTAS case management, persons enrolled at sites with co-located services had significantly higher rates of linkage to care at six months in a multivariate analysis [Compared with sites analysis . More imEstablishing working relationships and partnerships with local case management agencies enhanced the overall delivery and continuity of client services. At the majority of sites, the agency implementing the linkage program also provided Ryan White-funded case management services. Establishing the linkage program within an agency that provided Ryan White services allowed for a seamless transition for those clients who needed long-term case management upon discharge from short-term ARTAS case management. Regardless of whether or not the long-term case managers were located in-house, it was common for linkage staff to demonstrate how the program benefited case managers, especially emphasizing the preparatory work done by linkage staff  prior to the client's first meeting with the case manager.From an early stage of implementation, several project directors recognized that a change in job title was needed to reduce confusion and differentiate the roles and responsibilities of the short-term ARTAS linkage case manager from long-term or Ryan White case managers. Nearly all sites dropped the term "case manager" in favor of the term "linkage coordinator" or "linkage to care coordinator" to clarify that this position was transitional, short-term, and specific to facilitating linkage to medical care. Both clients and long-term case managers benefited from this distinction in terminology. In addition to making the linkage coordinator's role more transparent, another benefit of changing the job title was that it helped alleviate fears that the linkage program was competing for or "stealing" potential clients from long-term case managers. Dropping the term "case manager" from the job title helped mitigate territoriality or turf issues that arose when linkage coordinators were perceived as duplicating existing services rather than coordinating clients' entry into HIV medical care. Despite the clarification in job title, however, some Ryan White case managers expressed resentment over the discrepancy in caseload size (at agencies that housed both Ryan White case managers and linkage coordinators). To alleviate these concerns, supervisory staff scheduled periodic educational sessions with the case managers to ensure they had a thorough understanding of the linkage program and explain that smaller caseloads were needed due to the short-term, intensive nature of the intervention.Many ARTAS-II sites used similar methods to educate partners about the purpose and objectives of the linkage program and to delineate the roles and responsibilities of the linkage coordinator. Several sites held "lunch and learn" meetings to present information about the linkage to care program. Project directors at those sites noted that providing free lunch at these in-service sessions helped encourage attendance. Another common delivery method involved making special presentations to community partners, HIV consortia, and regional case management forums to educate stakeholders about the linkage to care program. Most sites created brochures and other informational materials about the linkage program to disseminate through meetings, in-services, and follow-up mailings. In Virginia, project staff recognized that their initial marketing materials were not effective because they were lengthy and convoluted, so they were replaced with simple, concise pocket cards or flyers that clearly outlined the benefits of the linkage program, which clients to refer, and who to contact. Virginia also created and presented case-based examples of the benefits of using a strengths-based approach to link clients to HIV medical care.Program staff consistently emphasized the benefits of the linkage to care program when communicating with potential referring partners and long-term case managers. As was mentioned earlier, linkage coordinators frequently completed intake assessments or eligibility paperwork, which reduced the overall workload for long-term case managers when clients were transitioned to them. Long-term case managers liked receiving clients who had eligibility and intake documentation completed because it freed them to do other work with clients. Another benefit of the program was that ARTAS clients were often better prepared than non-ARTAS clients when transitioned to long-term case management. Several project directors mentioned that the linkage coordinators took time to orient ARTAS clients to the case management system and the services that their case manager could provide. Lastly, the South Carolina sites marketed the linkage program by emphasizing the benefit to the patient's health and the overall public health in that patients who are in regular HIV medical care generally have better health  and are less likely to transmit HIV infection than those who are not in HIV care ,19.Across sites there was fairly uniform agreement about strategies that were most successful in generating and sustaining referrals to the linkage program. Maintaining ongoing communication and frequent contact (e.g. weekly) with supervisory and front-line staff at partner agencies was extremely important in ensuring the success of the referral process. In Virginia, program staff documented and monitored the number and types of contacts initiated by the linkage coordinators to establish new referral sources and partnerships. Documenting and tracking the number, frequency, and outcome of these contacts contributed to an increase in referrals throughout the project period. The Baton Rouge CBOs that implemented the linkage program hosted a quarterly networking lunch with their referring partners to provide updates on the progress of the linkage program and discuss successes and challenges with the referral process. This meeting also provided a forum in which community agencies could connect with each other and learn about the resources and services offered by each agency. Frequent communication with referral partners was crucial not only from a relationship-building perspective but also from a quality improvement perspective. Part of establishing and sustaining consistent referrals to the linkage program involved detecting problems or barriers with the existing referral process in order to make adjustments and improvements to the process. One of the most common issues that caused declines or total stoppage of referrals to the linkage program was staff turnover at referring agencies. When staff turnover occurred, the linkage coordinator had to work with the referral partner to identify a new person who would be responsible for making referrals to the linkage program and re-educate staff on the referral process.Another key strategy for sustaining referrals was providing referral partners with timely feedback on whether or not the clients they had referred had been linked to care. Providing referral partners with regular feedback was important in establishing trust and confidence in the linkage program and sustaining productive working relationships. In Virginia, DIS staff required formal documentation, such as a letter to place in their files, to confirm whether or not the client had been referred and linked to care. Less formal feedback mechanisms  were also used with referral partners to inform them about the client's linkage status. In Kansas City, linkage staff prepared and disseminated a quarterly newsletter that shared linkage "success stories", praised partners for successful referrals, and provided tips on making referrals to the linkage program. Referral partners were viewed as secondary clients, and providing feedback on referred clients was seen as good customer service. The feedback helped ensure that persons who made referrals received credit for their important role in connecting clients with HIV care.In addition to ongoing communication and providing referral partners with timely feedback, linkage coordinators also emphasized the importance of responding to all referrals in a timely and consistent manner. Timeliness of response to referrals was often facilitated by the proximity of the linkage coordinator to the referral source. At half of the program sites, the linkage coordinator was located in the same facility where HIV testing was conducted, or was available "on-call" by pager or cell phone to meet with the client in person immediately after the HIV-positive test result was delivered. On-call availability was highly valued by DIS staff in Alabama and Kansas City because the linkage coordinators could be contacted directly and they responded quickly, even assisting the client afterhours if needed. When it was not logistically feasible to meet with a client in person, the linkage coordinator attempted to call while the client was still with the HIV post-test counselor. The phone call provided an opportunity for the linkage coordinator to speak directly with the client, establish some initial rapport, and schedule a convenient time and place to meet with the client to discuss the linkage to care program. This initial, timely phone contact helped ensure that the client would eventually follow up and meet with the linkage coordinator. In Alabama and Kansas City, the linkage coordinators were trained and certified to conduct HIV testing, deliver test results, and provide pre- and post-test counseling. These sites found that coupling HIV counseling and testing with the primary duties of the linkage coordinator was an efficient use of staff time and resources and decreased the likelihood of client referrals falling through the cracks.One of the guiding strengths-based principles is making case management an active, community-based activity. Accordingly, many study sites allowed their linkage coordinators to be mobile and meet with referral partners and clients outside of the office setting . However, agency-specific policies prohibited some study sites from allowing their linkage coordinators to meet with clients outside of the office or transport clients in a personal or agency-owned vehicle. Nonetheless, the agencies that were allowed to do so found that the flexibility to meet with clients outside of the office setting was critical to the success of the linkage program. Meeting face-to-face with clients immediately after post-test counseling was extremely valuable to the linkage coordinator in building trust and establishing rapport with the client. At some sites, the linkage coordinator was able to use an agency-owned vehicle to meet with clients or referral partners outside of the office. At other sites, the linkage coordinator used a personal vehicle and was reimbursed for mileage. The linkage coordinators were usually covered under the agency's liability insurance for work conducted with clients outside of the office.A few sites even allowed their linkage coordinators to ride the bus or subway with clients to help them navigate the transportation system. Transportation is a common barrier to engaging in ongoing HIV medical care, and linkage coordinators used the strengths-based approach to help clients learn how to secure transportation and navigate their way to the clinic so they could become self-sufficient in arranging transportation in the future. The majority of sites also encouraged the linkage coordinator to meet clients at the HIV clinic and accompany them during their first HIV medical care visit. Allowing the linkage coordinators to be mobile and meet with clients outside of the office was vital to the success of the linkage program, especially among sites serving large, rural geographic areas where clients frequently had limited access to personal or public transportation.The short-term nature of the ARTAS linkage intervention requires a seamless process by which clients can transition from ARTAS case management to long-term case management, if needed--a process referred to as disengagement. Several techniques for handling the disengagement process were presented during the intervention training. The linkage coordinators were taught that disengagement is a graduated process that begins during the very first contact with the client and one that is reinforced during subsequent contacts. The client should be informed up front about the short-term, transitional nature and goals of ARTAS linkage case management. The linkage coordinator must ensure that clients fully understand that once they have completed five sessions together or have entered HIV primary care (whichever occurs first), they will be transferred to long-term case management services, if needed.The "active hand-off" is one model for facilitating the client's transition to long-term case management services provided through an HIV medical clinic or community agency. The active hand-off often involved introducing the client to his/her new case manager prior to disengagement and describing the types of case management services offered. Oftentimes, the linkage coordinator attended part of the client's first session with the new case manager. In Kansas City, the linkage coordinators used a statewide case management data capture system, so the comprehensive progress notes and any completed eligibility assessments were readily available to long-term case managers. Some linkage coordinators allowed clients to make a brief phone call after disengagement just to check in and let them know how things were going with their new case manager. The active hand-off model was especially useful when the linkage coordinator and long-term case managers were located at the same agency. Most importantly, clients had the opportunity to meet their long-term case manager prior to disengaging from the linkage coordinator. In addition, because of the intervention sessions with the linkage coordinator, most clients were already familiar with agency staff, agency culture and policies, and how to arrange transportation to the agency.One practice shared by the most successful implementing agencies was having a consistent, ongoing supervisory presence for the linkage coordinators. Ongoing supervision helps monitor the fidelity with which the linkage intervention is being delivered and ensures that the linkage coordinators adhere to strengths-based principles in their encounters with clients. Supervision was also critical for monitoring the disengagement process and supporting the linkage coordinators with the more difficult client cases. For all these reasons, it is important that the person supervising the linkage coordinators receive training in strengths-based methodology. The supervisor typically had a background in case management or social work and extensive experience with HIV service delivery systems. At a few sites, this person was responsible for supervising the linkage coordinators and the Ryan White case managers. The most common model of supervision was holding weekly case conference meetings during which client cases were discussed.Another level of supervision is needed when the state health department is overseeing implementation of the linkage program by community agencies. At this level, a staff person from the state HIV/AIDS program office needs to monitor implementation of the linkage program locally to ensure that the contracted agencies follow protocols and deliver linkage to care services as intended. For example, the linkage program directors at the state health departments in Louisiana, Virginia, and South Carolina monitored the number of referrals to the linkage program from various sources, gave quality assurance oversight, and provided programmatic or administrative support.As expected, when CDC funding ceased, 5 of the 10 study sites were unable to continue linkage to care activities. The five sites highlighted in this report fared better, with all five extending linkage activities in some capacity for at least several months beyond the end of project funding. However, these extensions were themselves dependent on small amounts of local funding which, in most cases, could not be sustained in the long run. South Carolina ceased all linkage activities when project funding ended, but re-started them several months later by incorporating linkage to care services in their CDC-funded Expanded HIV Testing Initiative project in emergency departments and in a Ryan White clinic using Minority AIDS Initiative funding. The effort to continue providing linkage services in South Carolina was successful because it used multiple funding streams  and employed one of the linkage coordinators from the ARTAS demonstration project. The Alabama CBO also used multiple funding sources to continue providing linkage services using one of the original linkage coordinators as a dual linkage/standard case manager. Louisiana was able to maintain one of the two linkage coordinators on a part-time basis for several months after the end of the project. The state HIV/AIDS programs in Missouri and Virginia were able to use Ryan White funding to continue supporting linkage program activities. Missouri also sponsored training opportunities in strengths-based methodology for case managers throughout the state.One key to setting the stage for future funding in Missouri was that the linkage program coordinator from Kansas City served on Missouri state committees for case management standards and quality improvement. She took advantage of any opportunity to report linkage to care program data, milestones, case studies, and anecdotal successes to the committee and to the state AIDS Director. In mid-2008, funding was expanded to quadruple the number of clients served by the linkage program in three areas of Missouri , which represent very different client populations across urban, suburban, and rural communities. The Kansas City, MO area has expanded the number of linkage coordinators, and has also done consulting work to expand the linkage program to the state of Kansas.Linkage to care is the important bridge between HIV counseling and testing and HIV medical care. Successful implementation of a linkage to care program requires attention to all the program practices described. As a group, sites that followed these best practices enrolled more participants, linked a higher percentage to medical care, and were able to continue linkage program activities for some period of time after CDC funding ended. It is noteworthy that the five grantees that continued the linkage program came from smaller metropolitan areas  compared to the five grantees that did not continue (average 4.1 million persons in 2000).Attention to these eight practices takes on additional urgency when HIV testing is not co-located with an onsite HIV clinic because inter-agency partnerships must be maintained. Strong inter-agency partnerships were a defining characteristic of sites that continued the linkage program after CDC funding ended. When multiple sources of newly diagnosed clients (across agencies) are involved, the organizational complexity to maintain these sources can be daunting. Nevertheless, implementing these best practices is not beyond the ability of even a small CBO. The five CBOs that were able to continue the linkage program after project funding ended had long-standing, cooperative relationships with local health departments, resulting in a steady influx of recently diagnosed HIV-positive persons. These CBOs recruited 56% of the 626 participants and recruited an average of 70 participants per grantee. In contrast, the three CBOs that did not continue the linkage program all experienced major challenges establishing and maintaining reliable relationships with local health departments. These CBOs recruited an average of 45 participants per grantee and averaged less than 70% linked to care.The strongest performing grantees were often, but not exclusively, those with co-located HIV medical care. As a group, co-located sites averaged 87% linkage to care compared to 73% for sites not co-located. Co-location of services makes initial entry to care easier for most clients. Yet there are many communities in the U.S. where co-location of an HIV clinic with one or more HIV testing sites is neither feasible nor sensible. The experience of these 10 implementing sites showed that even in communities where the linkage program was not co-located with an HIV clinic, achieving a linkage rate of 80% or higher was possible.Our report is not without limitations. We did not present statistical testing of the differences in linkage to care rates for site-level characteristics and best practices. Site-level best practices tend to be clustered, making separation of these characteristics on a small sample difficult. Modeling of these data that accounts for collinearity of site level factors and includes individual level factors was beyond the scope of this report.With the continuing push to expand offering of HIV testing in the United States, there is an urgent need to adapt linkage to care programs so that public health departments, community-based organizations, hospitals, emergency rooms, private doctors' offices, and HIV clinics are affiliated with a linkage to care program. To fully maximize the benefits of expanded HIV testing will require careful implementation, adaptation, and evaluation of linkage to care programs.The authors declare that they have no competing interests.JC was the study coordinator and participated in drafting the manuscript. LG conceived the study and participated in drafting the manuscript. AR managed the Kansas City site of the project and participated in editing the manuscript. DG managed the Baton Rouge site of the project and participated in editing the manuscript. NO managed the South Carolina site of the project and participated in editing the manuscript. DJ managed the Virginia site of the project and participated in editing the manuscript. RR provided the training for the linkage coordinators and participated in editing the manuscript. CS provided guidance to the design and implementation for the Alabama site of the project. KP managed the Alabama site of the project and participated in editing the manuscript.All authors read and approved the final manuscript.The pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/1472-6963/10/246/prepub


Twenty-nine laboratory-confirmed West Nile virus (WNV) encephalitis patients were bled serially so that WNV-reactive immunoglobulin (Ig) M activity could be determined. Of those patients bled, 7 (60%) of 12 had anti-WNV IgM at approximately 500 days after onset. Clinicians should be cautious when interpreting serologic results from early season WNV IgM-positive patients. In late summer and early fall of 1999, human West Nile virus (WNV) infections were recognized for the first time in the Western Hemisphere  M antibody capture enzyme-linked immunosorbent assay (MAC-ELISA), which demonstrates virus-reactive IgM in serum or cerebrospinal fluid (CSF) from a person with a clinically compatible illness   or A405nm (IgG ELISA) as described . An additional four specimens had equivocal results , which would require additional laboratory testing. In total, 18 (82%) of 22 specimens had either positive or equivocal results at approximately 200 days after onset.Scatter plot analysis of the IgM activity of all tested specimens showed a typical WNV-reactive IgM response , similar>3.0; range 3.1–6.5; average 4.0). An additional four specimens had equivocal results , which would require additional laboratory testing. In total, 13 (62%) of 21 specimens had either positive or equivocal results at 300–400 days after onset.Of the 21 serum specimens obtained approximately 300–400 days after onset, 9 (43%) were WNV-reactive IgM-positive  were WNV-reactive IgM-positive . An additional two specimens had equivocal results , which would require additional laboratory testing. In total, 7 (58%) of 12 specimens had either positive or equivocal results at approximately 500 days after onset, with the latest positive specimen having been drawn 525 days (17.5 months) after onset (P/N = 3.6).Of the 12 serum specimens obtained approximately As expected, most of the 29 case-patients had positive WNV-reactive IgG results  in their last tested serum specimen. The one patient whose P/N was <3.0 had a P/N of 2.8, which means that all specimens would probably be considered WNV antibody-positive or at least require additional laboratory testing. Although we did not follow longitudinally the WNV-reactive IgM activity in the CSF of these case-patients, the latest WNV-positive CSF specimen ever submitted to Centers for Disease Control and Prevention for diagnostic testing from a laboratory-confirmed WNV human infection was obtained at 47 days after onset (data not shown).The percent of patients with detectable IgM antibodies at 9 months differed by age , with 56Identification of a recent human infection with WNV is a important event that usually triggers public health alerts, mosquito control measures, and media attention. Because laboratory tests for the presence of WNV-reactive IgM are used to identify such infections, they should be conducted properly, and the test results must be interpreted accurately. Proper interpretation criteria include considering clinical context , previous travel history, flaviviral vaccination history, and evidence of previous and current WNV activity in the region. Cumulatively during 1999–2000, onset dates for human WNV disease cases in the United States ranged from early July to early December, roughly indicating a 5-month transmission season and a 7-month nontransmission season . Therefore, when evaluating a patient with acute viral encephalitis acquired in the United States, a positive serum test for IgM antibody to WNV would be expected to have a high predictive value, particularly during July to December, and especially when additional evidence exists of current epizootic or epidemic WNV activity in the area.., Florida), suspected cases of acute WNV disease of the central nervous system should be confirmed by the demonstration of WNV-reactive IgM in CSF, the development of WNV-specific IgG antibody in convalescent-phase serum , or both. We have determined empirically that the cross-reactivity of the WNV-reactive IgM appears to be less than that of Saint Louis encephalitis virus-reactive human IgM; therefore, conducting concurrent tests with the other indigenous neurotropic flaviviruses, which now coexist with WNV in some parts of the United States (Nevertheless, especially in areas where WNV is known to have circulated previously or has an extended transmission season (e.gThe observed apparent-to-inapparent WNV infection ratio in the United States is approximately 1:140, which indicates that a large group of persons with subclinical WNV infections exist (