Testes laboratoriais para peritonite infecciosa felina

Introdução – O que é PIF?

A peritonite infecciosa felina (PIF) é uma doença grave que afeta felinos domésticos e selvagens, cuja natureza é ubíqua (ou seja, ocorre em todo o mundo). O agente etiológico é o coronavírus felino (FCoV) que sofre mutação do biótipo entérico quase inofensivo (coronavírus entérico felino, FECV) para o biótipo sistêmico altamente virulento (vírus da peritonite infecciosa felina, FIPV) 1. O FCoV é um RNA vírus de fita simples, grande, envelopado e de polaridade positiva, comumente encontrado em gatos, com soroprevalência acima de 90% em lares com vários gatos 2. Este artigo oferece uma revisão da etiopatogenia do vírus e das opções de diagnóstico para PIF e, apesar de estar fora do escopo deste artigo, vale ressaltar que, embora a condição sempre tenha sido considerada inevitavelmente fatal, abordagens terapêuticas inovadoras (não aprovadas na maioria dos países) mostraram recentemente uma boa eficácia no tratamento da doença 3. 

Etiopatogenia da PIF

A transmissão viral é principalmente orofecal, e outras vias, como a salivar ou a transplacentária, são raramente descritas 1. Bandejas sanitárias representam a principal fonte de infecção, em que o FCoV pode sobreviver na matéria fecal por até 7 semanas 4. Os filhotes felinos costumam ser infectados quando os anticorpos maternos começam a declinar, em geral por volta de 5-6 semanas de idade 5. O FCoV então chega às células epiteliais colunares do intestino delgado, onde se replica e pode causar sinais gastrointestinais muito leves (ou, por vezes, mais graves) 6. Mesmo em gatos saudáveis, o vírus se replica dentro dos monócitos e, portanto, pode ser encontrado no sangue por um curto período de tempo 7. 

Foram identificados três padrões principais de eliminação do vírus na fezes. Uma pequena porcentagem de gatos (3-9%) parece ser resistente à infecção e nunca ou apenas brevemente elimina o vírus; 10-15% eliminarão o vírus a longo prazo ou de forma persistente, enquanto a maioria (70-80%) parece eliminar o vírus de maneira intermitente. Este último padrão é provavelmente uma consequência de reinfecções contínuas e/ou limitações dos testes de PCR 1,8. A excreção fecal em gatos jovens é muito alta, sobretudo em lares com vários gatos. Quanto maior a carga viral, maiores serão os níveis de replicação do vírus e, consequentemente, a taxa de mutação 8. Várias populações virais geneticamente aparentadas, mas distintas, se desenvolverão (quasispécies) e, em uma delas, ocorrerá uma mudança em seu tropismo celular para adquirir a capacidade de se replicar eficientemente no interior de monócitos/macrófagos e ativá-los e então se propagar de forma sistêmica 1,8.

Além disso, o tipo de resposta imune do hospedeiro, juntamente com fatores adicionais (por exemplo, estresse), pode desempenhar um papel tanto na patogênese como no tipo de doença 8. De fato, enquanto uma resposta mediada por células parece conferir resistência ao desenvolvimento da enfermidade, a forma “úmida” da PIF, caracterizada por efusões cavitárias, depende de uma resposta imune massiva mediada por linfócitos B. A forma não efusiva (seca) parece ser a consequência de uma resposta mediada por células parcialmente eficaz, o que confina as lesões a um número limitado de órgãos 9. É comum observar uma sobreposição entre as duas formas, em que casos não efusivos desenvolvem efusões nos estágios terminais ou as formas efusivas mostram lesões granulomatosas na necropsia 6. 

Embora seja amplamente aceito que a resposta imune pode influenciar o curso da infecção, ainda não foi identificada a mutação precisa (exata) que supostamente seja responsável pela mudança do biótipo FECV para FIPV. Isso limita a possibilidade de diagnosticar PIF por meio da identificação da cepa mutada, uma vez que os resultados da sorologia ou da técnica de PCR serão positivos em gatos infectados por qualquer um dos biótipos. Portanto, o diagnóstico deve ser formulado com base em vários outros achados clínicos e laboratoriais que podem fornecer resultados muito específicos ou aumentar a probabilidade diagnóstica de PIF 1,6,8.

Identificação e sinais clínicos

Os gatos com PIF costumam ser jovens (especialmente com menos de 2 anos), e os machos parecem ser mais suscetíveis. No entanto, animais mais idosos (com mais de 10 anos de idade) às vezes são acometidos, e os casos em gatos adultos aumentaram recentemente, sobretudo com a nova variante FCoV 23 8,10. De modo geral, há um histórico de evento estressante recente, como adoção ou castração 11. Animais de ambientes com vários gatos correm maior risco de desenvolver PIF; embora um estudo em grande escala tenha observado que a maioria dos gatos doentes eram de lares com um ou dois gatos, sugeriu-se que os gatos afetados haviam sido previamente expostos ao vírus 1,11. 

Os sinais clínicos comuns a ambas as formas da doença são letargia, inapetência, perda de peso/retardo de crescimento, febre (crescente e decrescente, 39,5-40°C), linfadenopatia e icterícia (Figura 1) 11,12. A PIF efusiva (úmida) é caracterizada por vasculite e serosite difusas, levando ao desenvolvimento de uma ou mais efusões cavitárias (abdominal, pleural, pericárdica e, raramente, escrotal), embora seja comum o relato de ascite e distensão abdominal (Figuras 2 e 3) 13. Os sinais de PIF não efusiva (seca) dependem da localização das lesões granulomatosas; em geral, essas lesões afetam o sistema nervoso central (que costumam se manifestar com convulsões, comportamento anormal, ataxia, nistagmo, hiperestesia ou, às vezes, paralisia e depressão), os olhos (muitas vezes, com uveíte e/ou coriorretinite) (Figura 4) e/ou os órgãos abdominais, como linfonodos, rins, fígado, baço e/ou trato gastrointestinal 1,8. Ocasionalmente, a PIF não efusiva pode ser localizada, com grandes massas abdominais palpáveis que podem se assemelhar a um tumor; essas massas podem ser causadas por aumento dos linfonodos mesentéricos ou lesões intestinais solitárias, especialmente do cólon ou da junção ileocecocólica (Figura 5) 13,14. 

Diagnosis of FIP 

Blood testing

Hematological changes indicative of an inflammatory process are commonly seen with FIP, but are nonspecific. The most frequent abnormalities are non-regenerative normocytic, normochromic anemia, lymphopenia, and neutrophilia with or without left shift. Microcytosis is also often observed, with or without anemia 11,13. 

Several biochemical abnormalities may be detected, which can be of some diagnostic accuracy. The protein profile usually shows hyperglobulinemia, with or without hyperproteinemia, low albumin concentration and low albumin-to-globulin ratio (A:G). FIP is considered very likely with a A:G < 0.4 and unlikely with a A:G > 0.8. Nevertheless, these values should be evaluated along with the clinical picture and other laboratory findings 8,13. 

Serum protein electrophoresis (SPE) will typically show decreased albumin, increased α2 fraction and a polyclonal gammopathy, although this latter can be less prominent if the SPE is performed early in the course of the disease (Figure 6) 15. 

Hyperbilirubinemia (in the absence of hemolysis, hepatic parenchymal alterations or cholestasis) is also commonly found, especially in the effusive form, and is probably a direct consequence of erythrocyte destruction within the lesions 8. Other biochemical alterations (e.g., increased liver enzyme levels) may be observed, depending on the localization and severity of the lesions 6. Most of the feline acute phase proteins (APPs), namely serum amyloid A, haptoglobin and α1-acid glycoprotein (AGP), increase greatly with FIP, but AGP is the most specific; in fact, marked increases in AGP support the diagnosis and can differentiate FIP from other inflammatory disorders 16,17. Nevertheless, again this has to be evaluated together with other alterations supportive of the disease.

As stated above, a positive result on serology will only demonstrate exposure to FCoV. Relatively higher antibody titers may be found in cats with FIP, but this can also occur in healthy cats in FCoV endemic catteries, due to continuous reinfection; at the same time a good percentage of FIP cats can be seronegative, due to antibodies forming complexes with circulating antigens 6. Similarly, viral RNA can be found in the blood of healthy animals. Additionally, the low viral load which is characteristic of FCoV viremia means that there is low analytical sensitivity on reverse transcription polymerase chain reaction (RT-PCR) testing. For these reasons, both serological and RT-PCR tests on blood should not be used as a diagnostic test for FIP 6,13.

Effusion tests 

FIP effusions have several peculiar features, and some tests will show optimal diagnostic accuracy, making sampling and analysis of effusions, when present, a necessary step. Macroscopically, the effusion is typically yellowish, sticky and can contain fibrin clots (Figure 7). Total protein content is high (> 3.5 g/dL) and cell count is typically low (< 5000 cell/µL), although this can be variable. Electrophoresis of the effusion will resemble that of SPE, and the A:G ratio is usually similarly low (< 0.4) 1,6,8. Cytology shows mostly non-degenerated neutrophils, macrophages and few lymphocytes on a granular eosinophilic proteinaceous background (Figure 8). Despite this non-specific pattern, cytology should always be performed to exclude the presence of a septic inflammatory process or neoplastic cells, which are other common causes of exudate that, if found, make FIP less likely 13.

Rivalta’s test is a cheap, point-of-care test performed by adding a drop of effusion to an acidic solution; a positive result is indicated if the effusion clots and retains its shape. This test has a high negative predictive value (i.e., a negative result makes FIP unlikely 18); however, a positive result alone cannot confirm FIP, since false positives are possible with other types of exudate (e.g., bacterial peritonitis, lymphoma). Again, using cytology together with this test will help identifying the disease 13,18,19. A similar test, the delta total nucleated cell count (DTNC), has been described utilizing a commercial hematology analyzer 20. Again, it relies on clumping of cells after addition of an acidic reagent, and involves using the analyzer to measure the leucocytes in two distinct channels. The DTNC – the ratio between the two obtained counts – is raised in FIP effusions, and has good diagnostic accuracy.

As with serum, the diagnostic accuracy of measuring antibody titers in effusions is low. FIP effusions can show negative serological results while having positive RT-PCR, sometimes even with an inverse correlation (i.e., negative serology despite a high viral load) 19. On the other hand, direct tests that demonstrate the antigen are very useful on effusions. RT-PCR shows good-to-very-good sensitivity and specificity, although false positives are sometimes observed 21. This can be secondary to circulating FCoV, even in small amounts, leaking from blood into the effusion due to inflammation of other origins. In general, a positive RT-PCR result coupled with cytological and biochemical alterations consistent with FIP is highly indicative of the disease 8.

Various immunocytochemistry (ICC) methods can be performed; these can demonstrate FCoV antigen inside macrophages via immunostaining, but the sensitivity is generally low to moderate, meaning that false negative results can easily occur; however, the specificity is high but not optimal, making it a good confirmatory test in the presence of other alterations supportive of the diagnosis 22. As with RT-PCR, false positive results can occur following viremia and leakage of viral RNA into the effusion of non-FIP cats, or from technical issues such as non-specific antibody binding 6,22.

Tests on other fluids

Neurological signs are more likely to occur in the non-effusive form of FIP, and cerebrospinal fluid (CSF) sampling may be appropriate in such cases. Clinicopathological alterations are non-specific but can be indicative of inflammation, such as increased protein content and mixed pleocytosis, usually pyogranulomatous. Nevertheless, cytology can also be unremarkable 8,19. Direct tests have proven utility, but with some limitations; as with effusions, ICC on CSF has shown high sensitivity, but the specificity is too low to make this a valuable confirmatory test 1,19.

RT-PCR has shown variably low to moderate sensitivity depending on the study, and a very good specificity (up to 100%), meaning that false positive results are very unlikely to occur. Additionally, the sensitivity of this test has been reported to increase drastically when only cats with neurological signs are evaluated, making RT-PCR on CSF a very useful test in these cases 8,19. Nevertheless, there is the rare chance that false positive results could result if the blood-brain barrier is damaged, causing leakage of circulating FCoV – demonstrating once again the importance of evaluating laboratory results together with other diagnostic methodologies supportive of the disease 19.

Aqueous humor (AH) can be collected in cats with ocular involvement (pyogranulomatous and granulomatous uveitis and/or chorioretinitis), with or without concomitant neurological signs 13. Clinicopathological changes have not been extensively described. Cytology can show neutrophilic, pyogranulomatous inflammation, and can be useful if neoplastic cells are present (e.g., lymphoma) 19. Total protein concentration is increased, especially in non-effusive FIP, and measurement can be useful (unpublished data).

Few studies have evaluated ICC for AH, and such studies show only moderate sensitivity and specificity, with false positive results recorded, so this option cannot be used as a confirmatory test. Further evaluation is needed to assess the diagnostic power of this test, since it could be a useful tool where other tests are not available (e.g., effusion analysis in non-effusive cases) 13. In addition, there are very few studies evaluating the use of RT-PCR on AH; reports show optimal specificity in the face of a very low sensitivity, making it a good tool to confirm the disease but not to exclude it, and again false positives are possible, when the blood-ocular barrier is damaged by other pathological processes (19; unpublished data).

Tissue testing

Currently, a definitive diagnosis of FIP is achieved by detection of typical histopathological changes in tissues, together with intralesional detection of FCoV using immunohistochemistry (IHC) (Figure 9) 19. Nevertheless, these tests have some limitations. Histological lesions indicative of FIP (i.e., pyogranuloma on serosal surfaces, granulomas, lymphoplasmacytic infiltrates, vasculitis) are not uniformly distributed, and can be missed on biopsy sampling. Viral antigen can also be variably distributed within lesions, thus multiple sections should be obtained in case of an unexpected negative IHC result. Moreover, the histological patterns typical of FIP can occasionally be observed with other conditions, making the use of IHC mandatory in these cases 23.

It is also noteworthy that laparoscopy or laparotomy to collect biopsy samples may be risky if the patient’s clinical condition is poor. Given this limitation, a few studies have evaluated the diagnostic accuracy of less invasive approaches. Cytological examination of affected organs has not been extensively studied, and specific changes have not been reported 8,24. ICC on fine-needle aspirates (FNAs) of liver and kidneys have low sensitivity, although a recent study evaluating FNAs on mesenteric lymph nodes showed reasonable sensitivity but a non-optimal specificity, with a false positive result probably related to the lymph nodes acting as a site of persistence of FCoV in non-FIP cats. Therefore, this test cannot exclude FIP, but can be a useful confirmatory test in conjunction with other consistent alterations 19,25.

When comparing results from IHC and RT-PCR on tissue biopsies, the former is more accurate, as RT-PCR has a lower specificity due to the well-recognized presence of systemic FCoV in non-FIP cats 8,23. On the other hand, RT-PCR on FNAs obtained from mesenteric nodes demonstrated a sensitivity and specificity of 90% and 96% respectively, with false negatives recorded in neurologic cases only. Therefore, this method can be a useful non-invasive addition to the diagnostic panel for FIP 24.

Detection of Spike gene mutations 

Although extensively studied for years, none of the mutations identified in sequences isolated from FIP cats have been shown to be specific for the disease 8. Mutations of the Spike (S) gene, which is responsible for host-receptor recognition and viral-cell-membrane fusion, have been evaluated in several studies, but the diagnostic power of this test is limited and is affected by the sequencing technique used. For example, a high number of false negative results will be recorded when some techniques (e.g., allelic discrimination) are used, since high viral loads are needed to obtain a result, and a failed sequencing is recorded as a negative result 6. On the other hands, when pyrosequencing is used, the specificity of the test is either low or no better than other techniques 21. Therefore, despite being likely involved in the pathogenesis of FIP, the variable detection of S gene mutations suggests that multiple mutations are probably involved, and its identification adds little or no information compared to conventional RT-PCR 8.

Conclusion

Feline infectious peritonitis is a worldwide disease, yet definitive diagnosis of the condition is elusive; antibody titers demonstrate only that a cat has been exposed to FCoV, and some FIP-affected cats can actually have negative titers. Hematology and biochemistry results can be helpful but not pathognomic for the disease, and testing on effusates can offer both false positive and false negative results. A combined approach using advanced diagnostic technologies is, therefore, recommended, and the importance of evaluating diverse laboratory results to reach a reasoned conclusion cannot be over-emphasized.