Evaluation of barcoded magnetic bead-based immunoassays for the simultaneous detection of feline leukemia virus antigen and antibody against feline immunodeficiency virus.

Testing platforms that leverage automation, require minimal sample volume, and enable various tests to be performed simultaneously on a single sample have the potential to improve workflow and efficiency in veterinary diagnostic laboratories. We evaluated a barcoded magnetic bead (BMB) technology using established immunoassays for detection of feline leukemia virus (FeLV) p27 antigen and antibody against feline immunodeficiency virus (FIV). Analytical sensitivity, limit of blank, and limit of detection were used to establish a functional sensitivity of 1.00 ng/mL of inactivated FeLV antigen and 35.7 ng/mL of anti-FIV monoclonal antibody. Common interferents, such as hemoglobin, lipid, and bilirubin, were not found to interfere with the performance of the assay. Intra- and inter-assay CVs were <13% for both assays using manufactured samples. Using a set of 116 feline samples, the diagnostic accuracy of our multiplex assay was 100% compared to reference assays. Performance in a convenience set of 1,000 feline samples submitted to a commercial diagnostic laboratory revealed a proportion of positive results of 1.3% for FeLV and 3.7% for FIV. BMB technology should enable rapid screening of samples for various markers in a single immunoassay well.

Feline immunodeficiency virus (FIV) infection in domestic pet cats in Australia and New Zealand: Guidelines for diagnosis, prevention and management.

Despite the passage of over 30 years since its discovery, the importance of feline immunodeficiency virus (FIV) on the health and longevity of infected domestic cats is hotly debated amongst feline experts. Notwithstanding the absence of good quality information, Australian and New Zealand (NZ) veterinarians should aim to minimise the exposure of cats to FIV. The most reliable way to achieve this goal is to recommend that all pet cats are kept exclusively indoors, or with secure outdoor access (e.g., cat enclosures, secure gardens), with FIV testing of any in-contact cats. All animal holding facilities should aim to individually house adult cats to limit the spread of FIV infection in groups of animals that are stressed and do not have established social hierarchies. Point-of-care (PoC) FIV antibody tests are available in Australia and NZ that can distinguish FIV-infected and uninfected FIV-vaccinated cats (Witness™ and Anigen Rapid™). Although testing of whole blood, serum or plasma remains the gold standard for FIV diagnosis, PoC testing using saliva may offer a welfare-friendly alternative in the future. PCR testing to detect FIV infection is not recommended as a screening procedure since a negative PCR result does not rule out FIV infection and is only recommended in specific scenarios. Australia and NZ are two of three countries where a dual subtype FIV vaccine (Fel-O-Vax® FIV) is available and offers a further avenue for disease prevention. Since FIV vaccination only has a reported field effectiveness of 56% in Australia, and possibly lower in NZ, FIV-vaccinated cats should undergo annual FIV testing prior to annual FIV re-vaccination using a suitable PoC kit to check infection has not occurred in the preceding year. With FIV-infected cats, clinicians should strive to be even more thorough than usual at detecting early signs of disease. The most effective way to enhance the quality of life and life expectancy of FIV-infected cats is to optimise basic husbandry and to treat any concurrent conditions early in the disease course. Currently, no available drugs are registered for the treatment of FIV infection. Critically, the euthanasia of healthy FIV-infected cats, and sick FIV-infected cats without appropriate clinical investigations, should not occur.

O diagnóstico do HIV/aids em homens heterossexuais: a surpresa permanece mesmo após mais de 30 anos de epidemia / HIV/AIDS diagnosis in heterosexual men: still a surprise after more than 30 years of the epidemic / El diagnóstico del VIH/SIDA en hombres heterosexuales: se mantiene la sorpresa incluso tras más de 30 años de epidemia

Resumo: Os homens são o principal grupo afetado pela infecção do HIV no Brasil, com tendência de crescimento nos últimos dez anos. Nos dados oficiais, os homens heterossexuais representam 49% dos casos, os homossexuais 38% e os bissexuais 9,1%. Os homens heterossexuais ficaram subsumidos na categoria de "população geral", não recebendo destaque em políticas ou ações de prevenção. O presente artigo se propõe a analisar as circunstâncias e estratégias por meio das quais os homens heterossexuais descobrem o diagnóstico do HIV. Busca-se, assim, compreender os caminhos percorridos, bem como os atores sociais envolvidos no diagnóstico de HIV/aids. Os dados analisados resultam de uma pesquisa qualitativa na qual foram entrevistados 36 homens vivendo com HIV/aids que não se identificam como homossexuais e/ou bissexuais. Esses homens foram contatados em três serviços especializados em aids de Porto Alegre, Rio Grande do Sul, Brasil. Os resultados indicam que eles se consideram imunes ao HIV, sendo o diagnóstico um evento inesperado. As mulheres (parceiras afetivo-sexuais e/ou ex-parceiras) são peças fundamentais para o diagnóstico masculino, pois revelam, seja pelo pré-natal, seja pelo adoecimento, a presença do HIV. Uma parcela importante dos homens se descobre soropositivo por ocasião de alguma doença, como a tuberculose, ou após várias idas e vindas dos serviços de saúde. A busca pela testagem de forma espontânea só acontece mediante a identificação de situações e sinais associados a uma possível contaminação. Os homens heterossexuais possuem poucas oportunidades de diagnóstico do HIV e, para além do gênero, são sujeitos à vulnerabilidade programática.

Abstract: Men are the main group affected by HIV infection in Brazil, with an upward trend in the last 10 years. According to official data, heterosexual men represent 49% of cases, followed by homosexuals with 38% and bisexuals with 9.1%. Heterosexual men have been subsumed in the category "overall population" and have failed to receive specific attention in preventive policies or activities. The article proposes to analyze the circumstances and strategies by which heterosexual men learn of their HIV diagnosis. The study thus seeks to understand the paths and social actors involved in their HIV/AIDS diagnosis. The data are from a qualitative study interviewing 36 men living with HIV/AIDS that did not self-identify as homosexuals and/or bisexuals. The men were contacted in three specialized AIDS services in Porto Alegre, Rio Grande do Sul State, Brazil. The results indicate that men consider themselves immune to HIV, and that the diagnosis is an unexpected event. Women (affective-sexual partners and/or former partners) are fundamental components in the men's diagnosis, since they reveal the presence of HIV through either prenatal care or their own illness. An important share of these men discover that they are HIV-positive through some illness such as tuberculosis or after several visits to health services. Spontaneous search for HIV testing only occurs through situations and signs associated with possible infection. Heterosexual men have few opportunities for HIV diagnosis, and beyond gender issues, they are subject to programmatic vulnerability.

Resumen: Los hombres son el principal grupo afectado por la infección del VIH en Brasil, con una tendencia de crecimiento en los últimos 10 años. En los datos oficiales, los hombres heterosexuales representan un 49% de los casos, los homosexuales un 38% y los bisexuales un 9,1%. Los hombres heterosexuales quedaron encajados en la categoría de "población general", no siendo relevantes en políticas o acciones de prevención. Este artículo se propone analizar las circunstancias y estrategias a través de las cuales los hombres heterosexuales descubren el diagnóstico del VIH. Se busca, de esta forma, comprender los caminos recorridos, así como los actores sociales implicados en el diagnóstico del VIH/SIDA. Los datos analizados son resultado de una investigación cualitativa en la que se entrevistaron a 36 hombres, viviendo con VIH/SIDA, que no se identifican como homosexuales y/o bisexuales. Se contactó con estos hombres a través de tres servicios especializados en sida de Porto Alegre, Rio Grande do Sul, Brasil. Los resultados indican que los hombres se consideran inmunes al VIH, siendo el diagnóstico un evento inesperado. Las mujeres (parejas afectivo-sexuales y/o ex-parejas) son piezas fundamentales para el diagnóstico masculino, puesto que revelan, sea a través del cuidado prenatal, sea a través de la enfermedad, la presencia del VIH. Una parte importante de los hombres se descubre seropositiva, debido a alguna enfermedad, como la tuberculosis, o tras varias idas y venidas a los servicios de salud. La búsqueda de un test espontáneo solamente se produce mediante la identificación de situaciones y señales asociadas a una posible infección. Los hombres heterosexuales poseen pocas oportunidades de diagnóstico del VIH y, más allá del género, están sujetos a vulnerabilidad programática.

Diagnosing feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV) infection: an update for clinicians.

With the commercial release in Australia in 2004 of a vaccine against feline immunodeficiency virus (FIV; Fel-O-Vax FIV®), the landscape for FIV diagnostics shifted substantially. Point-of-care (PoC) antibody detection kits, which had been the mainstay for diagnosing FIV infection since the early 1990s, were no longer considered accurate to use in FIV-vaccinated cats, because of the production of vaccine-induced antibodies that were considered indistinguishable from those produced in natural FIV infections. Consequently, attention shifted to alternative diagnostic methods such as nucleic acid detection. However, over the past 5 years we have published a series of studies emphasising that FIV PoC test kits vary in their methodology, resulting in differing accuracy in FIV-vaccinated cats. Importantly, we demonstrated that two commercially available FIV antibody test kits (Witness™ and Anigen Rapid™) were able to accurately distinguish between FIV-vaccinated and FIV-infected cats, concluding that testing with either kit offers an alternative to PCR testing. This review summarises pertinent findings from our work published in a variety of peer-reviewed research journals to inform veterinarians (particularly veterinarians in Australia, New Zealand and Japan, where the FIV vaccine is currently commercially available) about how the approach to the diagnosis of FIV infection has shifted. Included in this review is our work investigating the performance of three commercially available FIV PoC test kits in FIV-vaccinated cats and our recommendations for the diagnosis of FIV infection; the effect of primary FIV vaccination (three FIV vaccines, 4 weeks apart) on PoC test kit performance; our recommendations regarding annual testing of FIV-vaccinated cats to detect 'vaccine breakthroughs'; and the potential off-label use of saliva for the diagnosis of FIV infection using some FIV PoC test kits. We also investigated the accuracy of the same three brands of test kits for feline leukaemia virus (FeLV) diagnosis, using both blood and saliva as diagnostic specimens. Based on these results, we discuss our recommendations for confirmatory testing when veterinarians are presented with a positive FeLV PoC test kit result. Finally, we conclude with our results from the largest and most recent FIV and FeLV seroprevalence study conducted in Australia to date.

Epidemiological aspects of HIV infection and AIDS among indigenous populations / Aspectos epidemiológicos da infecção pelo HIV e da aids entre povos indígenas

ABSTRACT OBJECTIVE To describe the epidemiological aspects of HIV infection and AIDS among indigenous peoples of the state of Mato Grosso do Sul, Brazil. METHODS This is a descriptive epidemiological study on the occurrence and distribution of HIV infection and AIDS in the indigenous population assisted by the Distrito Sanitário Especial Indígena (Indigenous Special Health District) Mato Grosso do Sul between 2001 and 2014, based on three secondary databases. Annual rates of HIV and AIDS detection and prevalence were calculated, considering case distribution according to village, Health Base Pole and sociodemographic variables. Accumulated rates of detection, mortality and case fatality were calculated by ethnic group and for the Health Base Pole with the highest number of cases. RESULTS The HIV detection rate fluctuated between 0.0 and 18.0/100 thousand people in the study period. For AIDS, there was no notification before 2007, but in 2012 its rate reached 16.6/100 thousand. HIV prevalence grew between 2001 and 2011, and it continuously grew for AIDS starting from 2007. The highest HIV detection rates occurred among Guarani peoples (167.1/100 thousand) and for AIDS, among the Kaiowá peoples (79.3/100 thousand); mortality and fatality rates were higher among the Kaiowá. Regarding the Dourados Health Base Pole, the AIDS detection rate increased, and the mortality and fatality rates decreased. CONCLUSIONS HIV infection and AIDS have been increasing among indigenous peoples, with distribution of the disease mainly in the Health Base Poles of the southern region of the state, where greater economic and social vulnerability are also observed. The endemic character of HIV and AIDS can become epidemic in some years given the existence of cases in other villages in the state. Its occurrence among the Guarani and Kaiowá populations indicates the need for expanded diagnosis, access to treatment and prevention measures.

RESUMO OBJETIVO Descrever os aspectos epidemiológicos da infecção pelo HIV e da aids entre povos indígenas do Mato Grosso do Sul. MÉTODOS Estudo epidemiológico descritivo sobre ocorrência e distribuição da infecção pelo HIV e aids na população indígena assistida pelo Distrito Sanitário Especial Indígena Mato Grosso do Sul, entre 2001 e 2014, a partir de três bases de dados secundários. Calcularam-se as taxas anuais de detecção e de prevalência de HIV e aids, com distribuição dos casos segundo aldeia, Polo Base e variáveis sociodemográficas. As taxas acumuladas de detecção, mortalidade e letalidade foram calculadas por etnia e para os Polos Base com o maior número de casos. RESULTADOS A taxa de detecção de HIV flutuou entre 0,0 e 18,0/100 mil pessoas no período. Para a aids, não houve notificação antes de 2007, mas em 2012 sua taxa chegou a 16,6/100 mil. A prevalência de HIV indicou crescimento entre 2001 e 2011, e para a aids observou-se aumento contínuo a partir de 2007. As maiores taxas de detecção de HIV ocorreram entre os Guarani (167,1/100 mil) e de aids, entre os Kaiowá (79,3/100 mil); as taxas de mortalidade e letalidade foram superiores entre os Kaiowá. Para o Polo Base de Dourados, observou-se elevação da taxa de detecção de aids e diminuição das taxas de mortalidade e letalidade. CONCLUSÕES A infecção pelo HIV e a aids mostraram-se crescentes entre povos indígenas, com distribuição da doença principalmente nos Polos Base da região sul do estado, onde observa-se também maior vulnerabilidade econômica e social. O caráter endêmico do HIV e da aids pode se tornar epidêmico em alguns anos, considerando a existência de casos em outras aldeias do estado. Sua ocorrência entre os Guarani e Kaiowá sinaliza a necessidade de ampliação do diagnóstico, do acesso ao tratamento e de medidas de prevenção.

Concomitant feline immunodeficiency virus (FIV) and Mycoplasma haemofelis in a barn cat.

A 5-year-old male barn cat was presented with lethargy and excessive bleeding following castration. The patient developed hemolytic anemia and diagnostic tests revealed infection with feline immunodeficiency virus and Mycoplasma haemofelis. This case serves as a reminder of the importance of testing for infectious diseases and educating owners on feline infectious disease prevention and management.

Présence concomitante du virus de l'immunodéficience féline (FIV) et de Mycoplasma hæmofelis chez un chat de grange. Un chat de grange mâle âgé de 5 ans a été présenté avec de l'abattement et des saignements excessifs après la castration. Le patient a développé de l'anémie hémolytique et le diagnostic a révélé l'infection par le virus de l'immunodéficience féline et Mycoplasma hæmofelis. Ce cas peut servir de rappel de l'importance du dépistage de la présence de maladies infectieuses et de l'éducation des propriétaires sur la prévention et la gestion des maladies infectieuses félines.(Traduit par Isabelle Vallières).

Survival time and effect of selected predictor variables on survival in owned pet cats seropositive for feline immunodeficiency and leukemia virus attending a referral clinic in northern Italy.

Feline immunodeficiency virus (FIV) and feline leukemia virus (FeLV) are among the most important feline infectious diseases worldwide. This retrospective study investigated survival times and effects of selected predictor factors on survival time in a population of owned pet cats in Northern Italy testing positive for the presence of FIV antibodies and FeLV antigen. One hundred and three retrovirus-seropositive cats, 53 FIV-seropositive cats, 40 FeLV-seropositive cats, and 10 FIV+FeLV-seropositive cats were included in the study. A population of 103 retrovirus-seronegative age and sex-matched cats was selected. Survival time was calculated and compared between retrovirus-seronegative, FIV, FeLV and FIV+FeLV-seropositive cats using Kaplan-Meier survival analysis. Cox proportional-hazards regression analysis was used to study the effect of selected predictor factors (male gender, peripheral blood cytopenia as reduced red blood cells - RBC- count, leukopenia, neutropenia and lymphopenia, hypercreatininemia and reduced albumin to globulin ratio) on survival time in retrovirus-seropositive populations. Median survival times for seronegative cats, FIV, FeLV and FIV+FeLV-seropositive cats were 3960, 2040, 714 and 77days, respectively. Compared to retrovirus-seronegative cats median survival time was significantly lower (P<0.000) in FeLV and FIV+FeLV-seropositive cats. Median survival time in FeLV and FIV+FeLV-seropositive cats was also significant lower (P<0.000) when compared to FIV-seropositive cats. Hazard ratio of death in FeLV and FIV+FeLV-seropositive cats being respectively 3.4 and 7.4 times higher, in comparison to seronegative cats and 2.3 and 4.8 times higher in FeLV and FIV+FeLV-seropositive cats as compared to FIV-seropositive cats. A Cox proportional-hazards regression analysis showed that FIV and FeLV-seropositive cats with reduced RBC counts at time of diagnosis of seropositivity had significantly shorter survival times when compared to FIV and FeLV-seropositive cats with normal RBC counts at diagnosis. In summary, FIV-seropositive status did not significantly affect longevity of cats in this study, unlike FeLV and FIV+FeLV-seropositivity. Reduced RBC counts at time of FIV and FeLV diagnosis could impact negatively on the longevity of seropositive cats and therefore blood counts should always be evaluated at diagnosis and follow-up of retrovirus-seropositive cats.

Rapid and sensitive insulated isothermal PCR for point-of-need feline leukaemia virus detection.

Objectives Feline leukaemia virus (FeLV), a gamma retrovirus, causes diseases of the feline haematopoietic system that are invariably fatal. Rapid and accurate testing at the point-of-need (PON) supports prevention of virus spread and management of clinical disease. This study evaluated the performance of an insulated isothermal PCR (iiPCR) that detects proviral DNA, and a reverse transcription (RT)-iiPCR that detects both viral RNA and proviral DNA, for FeLV detection at the PON. Methods Mycoplasma haemofelis, feline coronavirus, feline herpesvirus, feline calicivirus and feline immunodeficiency virus were used to test analytical specificity. In vitro transcribed RNA, artificial plasmid, FeLV strain American Type Culture Collection VR-719 and a clinical FeLV isolate were used in the analytical sensitivity assays. A retrospective study including 116 clinical plasma and serum samples that had been tested with virus isolation, real-time PCR and ELISA, and a prospective study including 150 clinical plasma and serum samples were implemented to evaluate the clinical performances of the iiPCR-based methods for FeLV detection. Results Ninety-five percent assay limit of detection was calculated to be 16 RNA and five DNA copies for the RT-iiPCR, and six DNA copies for the iiPCR. Both reactions had analytical sensitivity comparable to a reference real-time PCR (qPCR) and did not detect five non-target feline pathogens. The clinical performance of the RT-iiPCR and iiPCR had 98.82% agreement (kappa[κ] = 0.97) and 100% agreement (κ = 1.0), respectively, with the qPCR (n = 85). The agreement between an automatic nucleic extraction/RT-iiPCR system and virus isolation to detect FeLV in plasma or serum was 95.69% (κ = 0.95) and 98.67% (κ = 0.85) in a retrospective (n = 116) and a prospective (n = 150) study, respectively. Conclusions and relevance These results suggested that both RT-iiPCR and iiPCR assays can serve as reliable tools for PON FeLV detection.

Performance of 4 Point-of-Care Screening Tests for Feline Leukemia Virus and Feline Immunodeficiency Virus.

BACKGROUND: More than 3 million cats in the United States are infected with FeLV or FIV. The cornerstone of control is identification and segregation of infected cats. HYPOTHESIS/OBJECTIVES: To compare test performance with well-characterized clinical samples of currently available FeLV antigen/FIV antibody combination test kits. ANIMALS: Surplus serum and plasma from diagnostic samples submitted by animal shelters, diagnostic laboratories, veterinary clinics, and cat research colonies. None of the cats had been vaccinated against FIV. The final sample set included 146 FeLV+, 154 FeLV-, 94 FIV+, and 97 FIV- samples. METHODS: Prospective, blind comparison to a gold standard: Samples were evaluated in 4 different point-of-care tests by ELISA antigen plate tests (FeLV) and virus isolation (FIV) as the reference standards. All test results were visually read by 2 blinded observers. RESULTS: Sensitivity and specificity, respectively, for FeLV were SNAP® (100%/100%), WITNESS® (89.0%/95.5%), Anigen® (91.8%/95.5%), and VetScan® (85.6%/85.7%). Sensitivity and specificity for FIV were SNAP® (97.9%/99.0%), WITNESS® (94.7%/100%), Anigen® (96.8%/99.0%), and VetScan® (91.5%/99.0%). CONCLUSIONS AND CLINICAL IMPORTANCE: The SNAP® test had the best performance for FeLV, but there were no significant differences for FIV. In typical cat populations with seroprevalence of 1-5%, a majority of positive results reported by most point-of-care test devices would be false-positives. This could result in unnecessary segregation or even euthanasia.

Viral diagnostic criteria for Feline immunodeficiency virus and Feline leukemia virus infections in domestic cats from Buenos Aires, Argentina.

A cross-sectional study was carried out on cats attending the Small Animal Hospital at the Faculty of Veterinary Sciences of the University of Buenos Aires to assess the prevalence and associated risk factors of Feline immunodeficiency virus (FIV) and Feline leukemia virus (FeLV) in the city of Buenos Aires, Argentina. Blood samples from 255 cats with symptoms compatible with FIV or FeLV infection, collected between 2009 and 2013 were analyzed by serology (immunochromatography, IA) and by hemi-nested PCR (n-PCR). The IA and n-PCR assays showed similar percentages of positivity for FIV while the n-PCR test was more sensitive for FeLV. Differences between the diagnostic tests and their choice according to the age of the animal are discussed. The clinical histories of ninety of the 255 cats showed blood profiles similar to others previously reported and revealed a higher risk of infection in male adult cats with outdoor access.

Diagnosing feline immunodeficiency virus (FIV) infection in FIV-vaccinated and FIV-unvaccinated cats using saliva.

We recently showed that two immunochromatography point-of-care FIV antibody test kits (Witness FeLV/FIV and Anigen Rapid FIV/FeLV) were able to correctly assign FIV infection status, irrespective of FIV vaccination history, using whole blood as the diagnostic specimen. A third FIV antibody test kit, SNAP FIV/FeLV Combo (an enzyme-linked immunosorbent assay [ELISA]), was unable to differentiate antibodies produced in response to FIV vaccination from those incited by FIV infection. The aim of this study was to determine if saliva is a suitable diagnostic specimen using the same well characterized feline cohort. FIV infection status of these cats had been determined previously using a combination of serology, polymerase chain reaction (PCR) testing and virus isolation. This final assignment was then compared to results obtained using saliva as the diagnostic specimen utilizing the same three point-of-care FIV antibody test kits and commercially available PCR assay (FIV RealPCR). In a population of cats where one third (117/356; 33%) were FIV-vaccinated, both immunochromatography test kits accurately diagnosed FIV infection using saliva via a centrifugation method, irrespective of FIV vaccination history. For FIV diagnosis using saliva, the specificity of Anigen Rapid FIV/FeLV and Witness FeLV/FIV was 100%, while the sensitivity of these kits was 96% and 92% respectively. SNAP FIV/FeLV Combo respectively. SNAP FIV/FeLV Combo had a specificity of 98% and sensitivity of 44%, while FIV RealPCR testing had a specificity of 100% and sensitivity of 72% using saliva. A revised direct method of saliva testing was trialed on a subset of FIV-infected cats (n=14), resulting in 14, 7 and 0 FIV positive results using Anigen Rapid FIV/FeLV, Witness FeLV/FIV and SNAP FIV/FeLV Combo, respectively. These results demonstrate that saliva can be used to diagnose FIV infection, irrespective of FIV vaccination history, using either a centrifugation method (Anigen Rapid FIV/FeLV and Witness FeLV/FIV) or a direct method (Anigen Rapid FIV/FeLV). Collection of a saliva specimen therefore provides an acceptable alternative to venipuncture (i) in fractious cats where saliva may be easier to obtain than whole blood, (ii) in settings when a veterinarian or trained technician is unavailable to collect blood and (iii) in shelters where FIV testing is undertaken prior to adoption but additional blood testing is not required.

Viral Reservoirs in Lymph Nodes of FIV-Infected Progressor and Long-Term Non-Progressor Cats during the Asymptomatic Phase.

BACKGROUND: Examination of a cohort of cats experimentally infected with feline immunodeficiency virus (FIV) for 5.75 years revealed detectable proviral DNA in peripheral blood mononuclear cells (PBMCs) harvested during the asymptomatic phase, undetectable plasma viral RNA (FIV gag), and rarely detectable cell-associated viral RNA. Despite apparent viral latency in peripheral CD4+ T cells, circulating CD4+ T cell numbers progressively declined in progressor animals. The aim of this study was to explore this dichotomy of peripheral blood viral latency in the face of progressive immunopathology. The viral replication status, cellular immunophenotypes, and histopathologic features were compared between popliteal lymph nodes (PLNs) and peripheral blood. Also, we identified and further characterized one of the FIV-infected cats identified as a long-term non-progressor (LTNP). RESULTS: PLN-derived leukocytes from FIV-infected cats during the chronic asymptomatic phase demonstrated active viral gag transcription and FIV protein translation as determined by real-time RT-PCR, Western blot and in situ immunohistochemistry, whereas viral RNA in blood leukocytes was either undetectable or intermittently detectable and viral protein was not detected. Active transcription of viral RNA was detectable in PLN-derived CD4+ and CD21+ leukocytes. Replication competent provirus was reactivated ex vivo from PLN-derived leukocytes from three of four FIV-infected cats. Progressor cats showed a persistent and dramatically decreased proportion and absolute count of CD4+ T cells in blood, and a decreased proportion of CD4+ T cells in PLNs. A single long-term non-progressor (LTNP) cat persistently demonstrated an absolute peripheral blood CD4+ T cell count indistinguishable from uninfected animals, a lower proviral load in unfractionated blood and PLN leukocytes, and very low amounts of viral RNA in the PLN. CONCLUSION: Collectively our data indicates that PLNs harbor important reservoirs of ongoing viral replication during the asymptomatic phase of infection, in spite of undetectable viral activity in peripheral blood. A thorough understanding of tissue-based lentiviral reservoirs is fundamental to medical interventions to eliminate virus or prolong the asymptomatic phase of FIV infection.

Efficacy of antiviral chemotherapy for retrovirus-infected cats: What does the current literature tell us?

GLOBAL IMPORTANCE: The two feline retroviruses, feline immunodeficiency virus (FIV) and feline leukaemia virus (FeLV), are global and widespread, but differ in their potential to cause disease. VIRAL INFECTION - FIV: FIV, a lentivirus that shares many properties with human immunodeficiency virus (HIV), can cause an acquired immune deficiency syndrome, which predisposes cats to other infections, stomatitis, neurological disorders and tumours. Although secondary infections are common, specific opportunistic infections or acquired immunodeficiency virus-defining infections, such as those that occur with HIV, are not commonly reported in FIV-infected cats. In most naturally infected cats, FIV does not cause a severe clinical syndrome; with appropriate care, FIV-infected cats can live many years before succumbing to conditions unrelated to their FIV infection. Thus, overall survival time is not necessarily shorter than in uninfected cats, and quality of life is usually high over many years or lifelong. VIRAL INFECTION - FELV: FeLV, an oncornavirus, is more pathogenic than FIV. Historically, it was considered to account for more disease-related deaths and clinical syndromes in cats than any other infectious agent. Recently, the prevalence and importance of FeLV have been decreasing, mainly because of testing and eradication programmes and the use of FeLV vaccines. Progressive FeLV infection can cause tumours, bone marrow suppression and immunosuppression, as well as neurological and other disorders, and leads to a decrease in life expectancy. However, with appropriate care, many FeLV-infected cats can also live several years with a good quality of life. PRACTICAL RELEVANCE: A decision regarding treatment or euthanasia should never be based solely on the presence or absence of a retrovirus infection. Antiviral chemotherapy is of increasing interest in veterinary medicine, but is still not used commonly. EVIDENCE BASE: This article reviews the current literature on antiviral chemotherapy in retrovirus-infected cats, focusing on drugs that are currently available on the market and, thus, could potentially be used in cats.

Determining the feline immunodeficiency virus (FIV) status of FIV-vaccinated cats using point-of-care antibody kits.

This study challenges the commonly held view that the feline immunodeficiency virus (FIV) infection status of FIV-vaccinated cats cannot be determined using point-of-care antibody test kits due to indistinguishable antibody production in FIV-vaccinated and naturally FIV-infected cats. The performance of three commercially available point-of-care antibody test kits was compared in a mixed population of FIV-vaccinated (n=119) and FIV-unvaccinated (n=239) cats in Australia. FIV infection status was assigned by considering the results of all antibody kits in concert with results from a commercially available PCR assay (FIV RealPCR™). Two lateral flow immunochromatography test kits (Witness FeLV/FIV; Anigen Rapid FIV/FeLV) had excellent overall sensitivity (100%; 100%) and specificity (98%; 100%) and could discern the true FIV infection status of cats, irrespective of FIV vaccination history. The lateral flow ELISA test kit (SNAP FIV/FeLV Combo) could not determine if antibodies detected were due to previous FIV vaccination, natural FIV infection, or both. The sensitivity and specificity of FIV RealPCR™ for detection of viral and proviral nucleic acid was 92% and 99%, respectively. These results will potentially change the way veterinary practitioners screen for FIV in jurisdictions where FIV vaccination is practiced, especially in shelter scenarios where the feasibility of mass screening is impacted by the cost of testing.

Rapid and sensitive detection of Feline immunodeficiency virus using an insulated isothermal PCR-based assay with a point-of-need PCR detection platform.

Feline immunodeficiency virus (FIV) is an important infectious agent of cats. Clinical syndromes resulting from FIV infection include immunodeficiency, opportunistic infections, and neoplasia. In our study, a 5' long terminal repeat/gag region-based reverse transcription insulated isothermal polymerase chain reaction (RT-iiPCR) was developed to amplify all known FIV strains to facilitate point-of-need FIV diagnosis. The RT-iiPCR method was applied in a point-of-need PCR detection platform--a field-deployable device capable of generating automatically interpreted RT-iiPCR results from nucleic acids within 1 hr. Limit of detection 95% of FIV RT-iiPCR was calculated to be 95 copies standard in vitro transcription RNA per reaction. Endpoint dilution studies with serial dilutions of an ATCC FIV type strain showed that the sensitivity of lyophilized FIV RT-iiPCR reagent was comparable to that of a reference nested PCR. The established reaction did not amplify any nontargeted feline pathogens, including Felid herpesvirus 1, feline coronavirus, Feline calicivirus, Feline leukemia virus, Mycoplasma haemofelis, and Chlamydophila felis. Based on analysis of 76 clinical samples (including blood and bone marrow) with the FIV RT-iiPCR, test sensitivity was 97.78% (44/45), specificity was 100.00% (31/31), and agreement was 98.65% (75/76), determined against a reference nested-PCR assay. A kappa value of 0.97 indicated excellent correlation between these 2 methods. The lyophilized FIV RT-iiPCR reagent, deployed on a user-friendly portable device, has potential utility for rapid and easy point-of-need detection of FIV in cats.

Diagnostic utility of CD4%:CD8 low% T-lymphocyte ratio to differentiate feline immunodeficiency virus (FIV)-infected from FIV-vaccinated cats.

Antibody testing based on individual risk assessments is recommended to determine feline immunodeficiency virus (FIV) status, but ELISA and Western blot tests cannot distinguish between anti-FIV antibodies produced in response to natural infection and those produced in response to FIV vaccination. The aim of this cross-sectional study was to test the hypothesis that FIV-infected cats could be differentiated from FIV-vaccinated uninfected cats using lymphocyte subset results, specifically the CD4%:CD8(low)% T-lymphocyte ratio. Comparisons of the CD4%:CD8(low)% T-lymphocyte ratio were made among the following four groups: Group 1 - FIV-infected cats (n=61; FIV-antibody positive by ELISA and FIV PCR positive); Group 2 - FIV-uninfected cats (n=96; FIV-antibody negative by ELISA); Group 3 - FIV-vaccinated uninfected cats (n=31; FIV-antibody negative by ELISA before being vaccinated against FIV, after which they tested FIV ELISA positive); and Group 4 - FIV-uninfected but under chronic/active antigenic stimulation (n=16; FIV-antibody negative by ELISA; all had active clinical signs of either upper respiratory tract disease or gingival disease for ≥ 21 days). The median CD4%:CD8(low)% T-lymphocyte ratio was lower in Group 1 (1.39) than in each of the other three groups (Group 2 - 9.77, Group 3 - 9.72, Group 4 - 5.64; P<0.05). The CD4%:CD8(low)% T-lymphocyte ratio was also the most effective discriminator between FIV-infected cats and the other three groups, and areas under ROC curves ranged from 0.91 (compared with Group 4) to 0.96 (compared with Group 3). CD4%:CD8(low)% shows promise as an effective test to differentiate between FIV-infected cats and FIV-vaccinated uninfected cats.

Domestic cat microsphere immunoassays: detection of antibodies during feline immunodeficiency virus infection.

Microsphere immunoassays (MIAs) allow rapid and accurate evaluation of multiple analytes simultaneously within a biological sample. Here we describe the development and validation of domestic cat-specific MIAs for a) the quantification of total IgG and IgA levels in plasma, and b) the detection of IgG and IgA antibodies to feline immunodeficiency virus (FIV) capsid (CA) and surface (SU) proteins, and feline CD134 in plasma. These assays were used to examine the temporal antibody response of domestic cats infected with apathogenic and pathogenic FIVs, and domestic cats infected with parental and chimeric FIVs of varying pathogenicity. The results from these studies demonstrated that a) total IgG antibodies increase over time after infection; b) α-CA and α-SU IgG antibodies are detectable between 9 and 28 days post-infection and increase over time, and these antibodies combined represent a fraction (1.8 to 21.8%) of the total IgG increase due to infection; c) measurable α-CD134 IgG antibody levels vary among individuals and over time, and are not strongly correlated with viral load; d) circulating IgA antibodies, in general, do not increase during the early stage of infection; and e) total IgG, and α-CA and α-SU IgG antibody kinetics and levels vary with FIV viral strain/pathogenicity. The MIAs described here could be used to screen domestic cats for FIV infection, and to evaluate the FIV-specific or total antibody response elicited by various FIV strains/other diseases.

Clinical aspects of feline retroviruses: a review.

Feline leukemia virus (FeLV) and feline immunodeficiency virus (FIV) are retroviruses with global impact on the health of domestic cats. The two viruses differ in their potential to cause disease. FeLV is more pathogenic, and was long considered to be responsible for more clinical syndromes than any other agent in cats. FeLV can cause tumors (mainly lymphoma), bone marrow suppression syndromes (mainly anemia), and lead to secondary infectious diseases caused by suppressive effects of the virus on bone marrow and the immune system. Today, FeLV is less commonly diagnosed than in the previous 20 years; prevalence has been decreasing in most countries. However, FeLV importance may be underestimated as it has been shown that regressively infected cats (that are negative in routinely used FeLV tests) also can develop clinical signs. FIV can cause an acquired immunodeficiency syndrome that increases the risk of opportunistic infections, neurological diseases, and tumors. In most naturally infected cats, however, FIV itself does not cause severe clinical signs, and FIV-infected cats may live many years without any health problems. This article provides a review of clinical syndromes in progressively and regressively FeLV-infected cats as well as in FIV-infected cats.

Validation of real-time polymerase chain reaction tests for diagnosing feline immunodeficiency virus infection in domestic cats using Bayesian latent class models.

The objectives of the current study were to estimate the sensitivity and specificity of three real-time polymerase chain reaction (PCR) tests for diagnosis of feline immunodeficiency virus (FIV) infection in domestic cats, both individually and when interpreted in series with one of two serological tests, separately in populations of cats at low and high risk of being infected with FIV. One PCR test targeted the pol gene and two targeted the gag gene of FIV. For comparison, sensitivities and specificities of the individual serological tests (IDEXX SNAP(®) test and AGEN Simplify(®) test) were also estimated. The study populations consisted of domestic cats thought to be not vaccinated against FIV. Low-risk (males aged 4 years or less and females; n=128) and high-risk (males over 4 years; n=128) cats were selected from those where blood samples were submitted to a commercial clinical pathology service. Bayesian latent class models were used to obtain posterior probability distributions for sensitivity and specificity for each test, based on prior distributions obtained from three experts. Medians of the posterior sensitivity distributions for the PCR tests based on the pol gene and two regions of the gag gene tests ranged from 0.85 to 0.89, compared to 0.89-0.97 for the two serological tests. The medians of posterior specificity distributions for these PCR tests were 0.94-0.96, and 0.95-0.97 for the serological tests. In contrast, the PCR based on one region of the gag gene had lower median sensitivity. Sensitivities of combinations of these serological and PCR tests interpreted in series were low; medians of posterior sensitivity distributions ranged from 0.75 to 0.83. Relative to the low-risk population, median sensitivities in the high-risk population were lower for all tests other than the AGEN Simplify(®) test; specificities were similar in both populations. We conclude that the sensitivities of the two PCR tests based on the pol gene and two regions of the gag gene, respectively, in non-vaccinated cats are probably lower than the sensitivities of the two serological tests we assessed. We do not recommend screening cats whose FIV vaccination status is uncertain with one of these serological tests and then testing positives with one of these PCR tests because in non-vaccinates, the sensitivities of combinations of these serological and PCR tests interpreted in series are low. Assessment of the validity of these PCR assays in FIV-vaccinated cats is required.