Low expression of antigen-presenting and costimulatory molecules by lung cells from tuberculosis patients

Costimulatory and antigen-presenting molecules are essential to the initiation of T cell immunity to mycobacteria. The present study analyzed by immunocytochemistry, using monoclonal antibodies and alkaline phosphatase-anti-alkaline phosphatase method, the frequency of costimulatory (CD86, CD40, CD40L, CD28, and CD152) and antigen-presenting (MHC class II and CD1) molecules expression on human lung cells recovered by sputum induction from tuberculosis (TB) patients (N = 22) and non-TB controls (N = 17). TB cases showed a statistically significant lower percentage of HLA-DR+ cells than control subjects (21.9 ± 4.2 vs 50.0 ± 7.2%, P < 0.001), even though similar proportions of TB cases (18/22) and control subjects (16/17, P = 0.36) had HLA-DR-positive-stained cells. In addition, fewer TB cases (10/22) compared to control subjects (16/17) possessed CD86-expressing cells (P = 0.04; OR: 0.05; 95%CI = 0.00-0.51), and TB cases expressed a lower percentage of CD86+ cells (P = 0.04). Moreover, TB patients with clinically limited disease (£1 lobe) on chest X-ray exhibited a lower percentage of CD86-bearing cells compared to patients with more extensive lung disease (>1 lobe) (P = 0.02). The lower expression by lung cells from TB patients of HLA-DR and CD86, molecules involved in antigen presentation and activation of T cells, may minimize T cell recognition of Mycobacterium tuberculosis, fostering an immune dysfunctional state and active TB.

Melatonin prevents inflammation and oxidative stress caused by abdominopelvic and total body irradiation of rat small intestine

We investigated the day-night differences in intestinal oxidative-injury and the inflammatory response following total body (TB) or abdominopelvic (AP) irradiation, and the influence of melatonin administration on tissue injury induced by radiation. Rats (male Wistar, weighing 220-280 g) in the irradiated groups were exposed to a dose of 8 Gy to the TB or AP region in the morning (resting period - 1 h after light onset) or evening (activity span - 13 h after light onset). Vehicle or melatonin was administered immediately before, immediately after and 24 h after irradiation (10, 2.0 and 10 mg/kg, ip, respectively) to the irradiated rats. AP (P < 0.05) and TB (P < 0.05) irradiation applied in the morning caused a significant increase in thiobarbituric acid reactive substance (TBARS) levels. Melatonin treatment in the morning (P < 0.05) or evening (P < 0.05) decreased TBARS levels after TB irradiation. After AP irradiation, melatonin treatment only in the morning caused a significant decrease in TBARS levels (P < 0.05). Although we have confirmed the development of inflammation after radiotherapy by histological findings, neither AP nor TB irradiation caused any marked changes in myeloperoxidase activity in the morning or evening. Our results indicate that oxidative damage is more prominent in rats receiving TB and AP irradiation in the morning and melatonin appears to have beneficial effects on oxidative damage irrespective of the time of administration. Increased neutrophil accumulation indicates that melatonin administration exerts a protective effect on AP irradiation-induced tissue oxidative injury, especially in the morning.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

Trypan blue exclusion assay by flow cytometry

Dye exclusion tests are used to determine the number of live and dead cells. These assays are based on the principle that intact plasma membranes in live cells exclude specific dyes, whereas dead cells do not. Although widely used, the trypan blue (TB) exclusion assay has limitations. The dye can be incorporated by live cells after a short exposure time, and personal reliability, related to the expertise of the analyst, can affect the results. We propose an alternative assay for evaluating cell viability that combines the TB exclusion test and the high sensitivity of the flow cytometry technique. Previous studies have demonstrated the ability of TB to emit fluorescence when complexed with proteins. According to our results, TB/bovine serum albumin and TB/cytoplasmic protein complexes emit fluorescence at 660 nm, which is detectable by flow cytometry using a 650-nm low-pass band filter. TB at 0.002% (w/v) was defined as the optimum concentration for distinguishing unstained living cells from fluorescent dead cells, and fluorescence emission was stable for 30 min after cell treatment. Although previous studies have shown that TB promotes green fluorescence quenching, TB at 0.002% did not interfere with green fluorescence in human live T-cells stained with anti-CD3/fluorescein isothiocyanate (FITC) monoclonal antibody. We observed a high correlation between the percentage of propidium iodide+CD3/FITC+ and TB+CD3/FITC+ cells, as well as similar double-stained cell profiles in flow cytometry dot-plot graphs. Taken together, the results indicate that a TB exclusion assay by flow cytometry can be employed as an alternative tool for quick and reliable cell viability analysis.

Single-nucleotide polymorphism analysis of GH, GHR, and IGF-1 genes in minipigs

Tibetan (TB) and Bama (BM) miniature pigs are two popular pig breeds that are used as experimental animals in China due to their small body size. Here, we analyzed single-nucleotide polymorphisms (SNPs) in gene fragments that are closely related to growth traits [growth hormone (GH), growth hormone receptor (GHR), and insulin-like growth factor (IGF)-1)] in these pig breeds and a large white (LW) control pig breed. On the basis of the analysis of 100 BMs, 108 TBs, and 50 LWs, the polymorphic distribution levels of GH, GHR, and IGF-1 were significantly different among these three pig breeds. According to correlation analyses between SNPs and five growth traits - body weight (BW), body length (BL), withers height (WH), chest circumference (CC), and abdomen circumference (AC) - three SNP loci in BMs and four SNP loci in TBs significantly affected growth traits. Three SNP sites in BMs and four SNP sites in TBs significantly affected growth traits. SNPs located in the GH gene fragment significantly affected BL and CC at locus 12 and BL at locus 45 in BMs, and also BW, WH, CC, and AC at locus 45 and WH and CC at locus 93 in TBs. One SNP at locus 85 in the BM GHR gene fragment significantly affected all growth traits. All indices were significantly reduced with a mixture of alleles at locus 85. These results provide more information regarding the genetic background of these minipig species and indicate useful selection markers for pig breeding programs.

Use of conventional PCR and smear microscopy to diagnose pulmonary tuberculosis in the Amazonian rainforest area

The diagnostic usefulness of Ziehl-Neelsen (ZN)-stained sputum smears combined with conventional polymerase chain reaction (ZN/PCR) to amplify IS6110 region DNA extracted from ZN slides was evaluated. The objective was to verify if this association could improve tuberculosis (TB) diagnosis in patients at remote sites. The study was carried out in 89 patients with culture-confirmed pulmonary TB as defined by the Brazilian Manual for TB Treatment. The participants were recruited in a reference unit for TB treatment in Rondônia, a state in the Amazonian area in northern Brazil. ZN, PCR, and culture performed in the sputum samples from these patients were analyzed in different combinations (i.e., ZN plus PCR and ZN plus culture). The prevalence rates of pulmonary TB in these patients were 32.6 and 28.1% considering culture and ZN/PCR, respectively. The sensitivity and specificity of ZN/PCR were 86 and 93%, respectively. ZN/PCR was able to detect more TB cases than ZN alone. This method could offer a new approach for accurate tuberculosis diagnosis, especially in remote regions of the world where culture is not available.

Development and applications of the SWAN rating scale for assessment of attention deficit hyperactivity disorder: a literature review

This study reviewed the use of the Strengths and Weaknesses of Attention-Deficit/Hyperactivity-symptoms and Normal-behaviors (SWAN) rating scale in diagnostic and evolutive approaches to attention deficit hyperactivity disorder (ADHD) and in correlational studies of the disorder. A review of articles published in indexed journals from electronic databases was conducted and 61 articles on the SWAN scale were analyzed. From these, 27 were selected to a) examine use of SWAN in research on attention disorders and b) verify evidence of its usefulness in the areas of genetics, neuropsychology, diagnostics, psychiatric comorbidities, neuroimaging, pharmacotherapy, and to examine its statistical reliability and validity in studies of diverse populations. This review of articles indicated a growing use of the SWAN scale for diagnostic purposes, for therapy, and in research on areas other than ADHD, especially when compared with other reliable scales. Use of the scale in ADHD diagnosis requires further statistical testing to define its psychometric properties.

Efeito da temperatura de extrusão na absorção de água, solubilidade e dispersibilidade da farinha pré-cozida de milho-soja (70:30)

Com o objetivo de verificar a possibilidade do uso das farinhas pré-cozidas de milho-soja (70:30) por extrusão em produtos alimentícios, foram estudadas suas características tecnológicas. A mistura crua de milho e soja foi extrusada num extrusor Brabender de dupla-rosca com velocidade de alimentação constante de 20,8Kg/h, velocidade da rotação de parafuso de 100rpm e diâmetro de matriz de 5mm. Os perfis da temperatura de barril (TB) foram: zona 1,60ºC (constante); zona 2,90ºC (constante); zona 3, 90, 110, 130 ou 150ºC e zona 4 (matriz), 110, 130 ou 150ºC. Os resultados mostraram que, o aumento da TB nas zonas 3 e 4 causou um aumento na absorção de água, nitrogênio solúvel em água, índice de solubilidade de nitrogênio, proteína dispersível em água e índice de dispersibilidade de proteína. Não foi verificada a atividade do inibidor de tripsina nas farinhas extrusadas estudadas. Sugere-se, portanto, o uso destas nos produtos cárneos e de panificação.

Spectrally Tuned Lanthanide Photoluminescence for Point-of-Care Testing

Point-of-care (POC) –diagnostics is a field with rapidly growing market share. As these applications become more widely used, there is an increasing pressure to improve their performance to match the one of a central laboratory tests. Lanthanide luminescence has been widely utilized in diagnostics because of the numerous advantages gained by the utilization of time-resolved or anti-Stokes detection. So far the use of lanthanide labels in POC has been scarce due to limitations set by the instrumentation required for their detection and the shortcomings, e.g. low brightness, of these labels. Along with the advances in the research of lanthanide luminescence, and in the field of semiconductors, these materials are becoming a feasible alternative for the signal generation also in the future POC assays. The aim of this thesis was to explore ways of utilizing time-resolved detection or anti-Stokes detection in POC applications. The long-lived fluorescence for the time-resolved measurement can be produced with lanthanide chelates. The ultraviolet (UV) excitation required by these chelates is cumbersome to produce with POC compatible fluorescence readers. In this thesis the use of a novel light-harvesting ligand was studied. This molecule can be used to excite Eu(III)-ions at wavelengths extending up to visible part of the spectrum. An enhancement solution based on this ligand showed a good performance in a proof-of-concept -bioaffinity assay and produced a bright signal upon 365 nm excitation thanks to the high molar absorptivity of the chelate. These features are crucial when developing miniaturized readers for the time-resolved detection of fluorescence. Upconverting phosphors (UCPs) were studied as an internal light source in glucose-sensing dry chemistry test strips and ways of utilizing their various emission wavelengths and near-infrared excitation were explored. The use of nanosized NaYF :Yb3+,Tm3+-particles enabled the replacement of an external UV-light source with a NIR-laser and gave an additional degree of freedom in the optical setup of the detector instrument. The new method enabled a blood glucose measurement with results comparable to a current standard method of measuring reflectance. Microsized visible emitting UCPs were used in a similar manner, but with a broad absorbing indicator compound filtering the excitation and emission wavelengths of the UCP. This approach resulted in a novel way of benefitting from the non-linear relationship between the excitation power and emission intensity of the UCPs, and enabled the amplification of the signal response from the indicator dye.

Farinha de trigo e soja pré-cozida por extrusão para uso em croquete de carne

Com o objetivo de obter uma farinha pré-cozida de trigo e soja (90:10) com boas características tecnológicas para uso em croquete de carne, foram estudados efeitos de parâmetros da extrusão na capacidade emulsificante (CE) e na estabilidade de emulsão (EE) das farinhas obtidas, e nas características sensoriais do produto elaborado. A mistura foi extrusada em diferentes umidades (23, 26 e 29%), temperaturas de barril (TB: 100, 110, 120 e 130 °C) e velocidades de rotação de parafuso (VRP: nº 4; 120, 150, 180 e 210 rpm). Os resultados indicam que o aumento de VRP e TB aumentou CE e EE até certo ponto, que, ao ser ultrapassado, as fez diminuir. O croquete de carne elaborado com farinha mista com 26% de umidade e extrusada em 150 rpm a 130 °C mostrou melhores sabor e textura, sendo o preferido pela equipe massal de provadores não treinados ao compará-lo com aqueles da farinha de trigo crua e da farinha mista crua. As características sensoriais dos croquetes estudados foram relacionadas com CE e EE das farinhas usadas, sendo que a EE mostrou um efeito mais acentuado do que a CE.

Pellets de trigo e soja produzidos por extrusão

A mistura de trigo e soja representa uma importante fonte calórico-protéica com proteínas de boa qualidade. Apesar disso, a utilização da soja como ingrediente tem sido limitada pelo seu sabor de feijão cru (beany flavor), o qual é melhorado pelo processo de extrusão. Foram estudados os efeitos de umidade da mistura, Temperatura de Barril (TB) e Velocidade de Rotação de Parafuso (VRP, Nº 5) do extrusor no Índice de Expansão (IE), no Índice de Solubilidade em Água (ISA) e nas características sensoriais de pellets produzidos com mistura de trigo e soja (90:10), objetivando a otimização deste processo para a obtenção de pellets fritos com melhores qualidades sensoriais. A farinha mista crua foi extrusada em 2 umidades (32 e 35%), 4 TB (60 a 90 °C) e 4 VRP (60 a 150 rpm), totalizando 32 tratamentos. O pellet frito, preparado com a farinha mista com 32% de umidade e extrusada em 60 rpm a 80 °C, apresentou o maior IE e as melhores qualidades sensoriais (aparência, sabor e textura), sendo preferido pela equipe de provadores não treinados, tanto com sabor de bacon como com sabor de queijo.

Prevalência de tuberculose em transplantados renais: revisão sistemática e meta-análise

INTRODUÇÃO: A prevalência de tuberculose entre transplantados renais (TB-TXR) é maior do que na população geral. Assim, objetivamos realizar uma revisão sistemática e meta-análise da prevalência de TB-TXR. MÉTODOS: Após buscas eletrônicas e revisão de referências, estimou-se a prevalência agrupada de TB-TXR e realizou-se meta-regressão. Como referência para comparações, utilizou-se a prevalência de TB na população geral (0,18%; 95% IC = 0,16-0,20). RESULTADOS: Foram triados 253 estudos e 41 incluídos. A prevalência agrupada de TB-TXR foi 2,51% (95% IC = 2,17-2,85). Na meta-regressão, amostra > 2501 e alta prevalência de TB na população geral (p < 0,05) permaneceram associadas com a prevalência de TB-TXR. CONCLUSÃO: A prevalência agrupada de TB-TXR encontrada foi 14 vezes maior do que a prevalência de TB na população geral e, dessa forma, destacamos a necessidade de que o planejamento de medidas de prevenção e controle da TB específicas para este grupo de indivíduos seja pauta nas discussões do setor saúde.

Blue and Uv-Emitting Upconversion Nanoparticles: Synthesis and (Bio) Analytical Applications.

Rare-earth based upconverting nanoparticles (UCNPs) have attracted much attention due to their unique luminescent properties. The ability to convert multiple photons of lower energy to ones with higher energy through an upconversion (UC) process offers a wide range of applications for UCNPs. The emission intensities and wavelengths of UCNPs are important performance characteristics, which determine the appropriate applications. However, insufficient intensities still limit the use of UCNPs; especially the efficient emission of blue and ultraviolet (UV) light via upconversion remains challenging, as these events require three or more near-infrared (NIR) photons. The aim of the study was to enhance the blue and UV upconversion emission intensities of Tm3+ doped NaYF4 nanoparticles and to demonstrate their utility in in vitro diagnostics. As the distance between the sensitizer and the activator significantly affect the energy transfer efficiency, different strategies were explored to change the local symmetry around the doped lanthanides. One important strategy is the intentional co-doping of active (participate in energy transfer) or passive (do not participate in energy transfer) impurities into the host matrix. The roles of doped passive impurities (K+ and Sc3+) in enhancing the blue and UV upconversions, as well as in influencing the intense UV upconversion emission through excess sensitization (active impurity) were studied. Additionally, the effects of both active and passive impurity doping on the morphological and optical performance of UCNPs were investigated. The applicability of UV emitting UCNPs as an internal light source for glucose sensing in a dry chemistry test strip was demonstrated. The measurements were in agreement with the traditional method based on reflectance measurements using an external UV light source. The use of UCNPs in the glucose test strip offers an alternative detection method with advantages such as control signals for minimizing errors and high penetration of the NIR excitation through the blood sample, which gives more freedom for designing the optical setup. In bioimaging, the excitation of the UCNPs in the transparent IR region of the tissue permits measurements, which are free of background fluorescence and have a high signal-to-background ratio. In addition, the narrow emission bandwidth of the UCNPs enables multiplexed detections. An array-in-well immunoassay was developed using two different UC emission colours. The differentiation between different viral infections and the classification of antibody responses were achieved based on both the position and colour of the signal. The study demonstrates the potential of spectral and spatial multiplexing in the imaging based array-in-well assays.

Biomarkkerit sepsiksen diagnostiikassa sekä Kvantitatiivisen prokalsitoniini-immunomäärityksen pystyttäminen ja optimointi point-of-care-ympäristöön

Termiä sepsis käytetään infektion aiheuttamasta yleisestä tulehdusreaktio-oireyhtymästä. Biokemiallinen merkkiaine eli biomarkkeri on aine, joka objektiivisesti mitattuna ja arvioituna toimii indikaattorina erottaen toisistaan normaalit biologiset prosessit ja patogeeniset prosessit. Lisäksi biomarkkeri saattaa osoittaa farmakologisen vasteen lääkehoidolle. Tutkielman kirjallisessa osassa on esitelty ja verrattu toisiinsa jo kliinisessä käytössä olevia sekä lupaavia, vielä tutkimusvaiheessa olevia, sepsiksen biomerkkiaineita. Lisäksi tutkielmassa on tarkasteltu sepsiksen todentamisen, tunnistamisen ja hoidon tulevaisuudennäkymiä. Prokalsitoniini (PCT) on eräs jo kliinisessä käytössä oleva sepsiksen biomerkkiaine. Tutkielman kokeellisessa osassa pystytettiin teho-osastoille suunnattu pikatesti PCT:n mittaamiseen aikaerotteista fluoresenssiteknologiaa hyödyntävälle AQT-järjestelmälle (DHR Finland Oy, Innotrac Diagnostics, Suomi). Järjestelmä mittaa 20–30 minuutissa biomerkkiainepitoisuuden. Tavoitteena oli löytää sopiva vasta-ainepari ja pystyttää kaksipuoleinen immunomääritys, jonka analyyttinen herkkyys on alle 0,02 ng/ml:ssa. Työssä tutkittiin biotinyloidun kokonaisen vasta-aineen ja paikkaspesifisesti biotinyloidun antigeenia sitovan fragmentin (engl. fragment antigen binding, Fab) toimivuutta sitojamolekyylinä. Lisäksi työssä verrattiin kahden eri leimamolekyylin, 9 hampaisen europiumkelaatin sekä Innotracissa kehitetyn tähtikelaatin, toimivuutta PCT-immunomäärityksessä. Vasta-aineiden mahdolliset ristireaktiot testattiin ja määritystä testattiin alustavasti eri näytematriiseissa. Työssä löydettiin 5 vasta-aineparia, joiden analyyttinen herkkyys oli alle 0,02 ng/ml:ssa. Aiemmin testattuja vasta-ainepareja parempi analyyttinen herkkyys johtui todennäköisesti yhden uuden leimavasta-aineen paremmasta affiniteetistä. Tähtikelaatilla leimatulla vasta-aineella analyyttinen herkkyys parani lähes viisinkertaisesti, kun sitä verrattiin 9-hampaisella euroopiumkelaatilla leimatun vasta-aineen määritykseen. Havaittiin myös, että Fab-fragmentilla mitattiin määrityksissä lähes kaksi kertaa suuremmat signaali-taustasuhteet, kun sitä verrattiin kokonaiseen vasta-aineeseen, tietyllä vasta-aineparilla. Tämä johtui luultavasti steeristen esteiden vähenemisestä. Näytematriisilla ei todettu olevan vaikutusta määritykseen, eikä ristireaktioita havaittu. Kehitetyn määrityksen suoriutumista verrattiin markkinoilla olevaan BRAHMS AG:n (Saksa) PCT-määritykseen käyttäen potilasnäytteitä. Korrelaatiokertoimet potilasnäytevertailuissa olivat 0,965–0,989 ja näytteiden mitatuissa pitoisuuksissa oli tasoero.

Epstein-Barr-viruksen antigeeniosoitustestin kehittäminen

Epstein-Barr-virus (EBV) aiheuttaa mononukleoosia eli pusutautia, joka ilmenee yleensä murrosiällä tai nuorella aikuisiällä. Mononukleoosissa on tyypilliset nielutulehduksen oireet, minkä takia sitä on vaikea erottaa muiden taudinaiheuttajien aiheuttamista nielutulehduksista. Erotusdiagnostiikan käyttö nielutulehduksessa on oleellista, koska vain Streptokokki-bakteerien aiheuttamat nielutulehdukset vaativat antibioottihoitoa. Akuutin EBV-infektion pikadiagnostiikka perustuu nykyisellään infektion seurauksena muodostuvien heterofiilisten vasta-aineiden mittaamiseen verestä. Niiden mittaamisessa on useita ongelmia, koska useilla lapsilla niitä ei muodostu lainkaan ja vanhemmillakin niitä muodostuu yleensä vasta viikon päästä mononukleoosin oireiden alkamisesta. Mittaamalla EBV:n antigeeneja saataisiin positiivinen testitulos vasta-ainetestiä nopeammin. EBV:lle ei ole kuitenkaan kehitetty antigeeniosoitustestiä todennäköisesti siksi, että EBV:n erittymisen terveiden viruksen kantajien limakalvoille uskotaan olevan ongelma antigeenitestauksessa. Diplomityön tavoitteena oli kehittää akuutin EBV-infektion pikadiagnostiikkaan soveltuva limakalvonäytteestä tehtävä immunometrinen antigeeniosoitustesti. Työssä kehitettiin uusia polyklonaalisia vasta-aineita sekä testattiin kaupallisia vasta-aineita. Vasta-aineiden toimintaa tutkittiin immunomäärityksissä kokonaista EBV:ta ja puhdistettuja proteiiniantigeeneja vastaan. Monoklonaalisten vasta-aineiden kehitys lopetettiin ennen varsinaista tuottoa, koska ensin kehitetyt polyklonaaliset vasta-aineet eivät tunnistaneet natiivia virusta vaan pelkästään immunisointeihin käytetyt rekombinanttiset kohdeproteiinit. Kaupallisista vasta-aineista yhdellä onnistuttiin kehittämään tiettävästi maailman ensimmäinen immunometrinen EBV-antigeeniosoitustesti, jossa saatiin tunnistus sekä natiivilla EBV:lla että puhdistetulla proteiiniantigeenilla. Testin herkkyys proteiiniantigeenilla oli hyvä (4 pM) ja puhdistetulla EBV-virusvalmisteellakin todennäköisesti riittävä (6,2×106 viruspartikkelia/ml) kliiniseen diagnostiikkaan. Testillä ei kuitenkaan saatu suppeasta mononukleoosipotilaiden nielunäyteaineistosta yhtään EBV-positiivista tulosta. Referenssiksi tilatut PCR-testit osoittivat näytteiden EBV-pitoisuuksien olevan liian alhaisia osoitettavaksi kehitetyllä EBV-antigeeniosoitustestillä. PCR-testauksessa mononukleoosipotilaiden nielunäytteistä osoitettujen alhaisten EBV-määrien perusteella spekuloitiin, että olisiko edustavin näytteenottopaikka akuutissa EBV-infektiossa sittenkin nielun sijaan nenänielu. Myös kirjallisuudesta löytyi tälle tukea. EBV-antigeeniosoitustestin osoitettiin toimivan hyvin standardinäyte-materiaalilla. Testikehitysprojektin jatkon kannalta oleellista on kuitenkin vielä selvittää laajemmalla kliinisellä näyteaineistolla, mistä EBV:n antigeeneja pitäisi osoittaa akuutissa EBV-infektiossa ja ovatko niiden pitoisuudet riittävän korkeita.