ABSTRACT A study with transmission electron microscopy of mycoplasma-contaminated HeLa cells using five cell donors referred to as donors A, B, C, D and E, observations are herein presented. Experiments performed with cells from donors B, C and D, revealed the presence of Mycoplasma hyorhinis after PCR and sequencing experiments. Bacteria probably originated from a cytoplasm with compacted tiny granular particles replacing the normal cytosol territories, or from the contact with the cytoplasm through a clear semi-solid material. The compact granularity (CG) of the cytoplasm was crossed by stripes of smooth and rough endoplasmic reticulum cisternae. Among apparently normal mitochondria, it was noted, in variable proportions, mitochondria with crista-delimited lucent central regions that expand to and occupied the interior of a crista-less organelle, which can undergo fission. Other components of the scenarios of mycoplasma-induced cell demolition are villus-like structures with associated 80-200 nm vesicles and a clear, flexible semi-solid, process-sensitive substance that we named jam-like material. This material coated the cytoplasmic surface, its recesses, irregular protrusions and detached cytoplasmic fragments. It also cushioned forming bacteria. Cyst-like structures were often present in the cytoplasm. Cells, mainly apoptotic, exhibiting ample cytoplasmic sectors with characteristic net-like profile due to adjoined vacuoles, as well as ovoid or elongated profiles, consistently appeared in all cells from the last four cell donors. These cells were named “modified host cells” because bacteria arose in the vacuoles. The possibility that, in some samples, there was infection and/or coinfection of the host cell by another organism(s) cannot be ruled out.

A aplicação clínica dos antagonistas de CCR5 envolve em primeiro lugar determinar o uso de co-receptor pela cepa viral infectante. Programas de bioinformática que prevêem o uso co-receptor poderiam fornecer um método alternativo para selecionar candidatos para o tratamento com os antagonistas do CCR5, particularmente em países com poucos recursos financeiros. Assim, o presente estudo teve por objetivo identificar a melhor abordagem utilizando ferramentas de bioinformática para determinar qual o tipo de co-receptor do HIV-1 que poderia ser usado na prática clínica. Sequências de DNA proviral e Trofile resultados a partir de 99 pacientes infectados pelo HIV-1 sob monitorização clínica foram avaliadas. Com base nos resultados do Teste Trofile, as variantes virais presentes eram R5 (81,1%), R5X4 (21,4%) e X4 (1,8%). Determinação do tropismo pela análise do Geno2pheno, com taxa de falso positivos de 10% apresentou desempenho mais adequado para esta amostragem: as cepas R5 e X4 foram encontradas em frequências de 78,5% e 28,4%, respectivamente, e foi de 78,6% a concordância entre os resultados fenotípicos e genotípicos. Mais estudos são necessários para esclarecer como a diversidade genética entre as cepas do vírus afeta abordagens baseadas na determinação do tropismo pelas ferramentas de bioinformática. Embora esta estratégia possa ser útil para o rastreio de pacientes em países em desenvolvimento, permanecem algumas limitações que restringem a aplicação mais ampla para utilização de testes de co-receptor na prática clínica.

The clinical application of CCR5 antagonists involves first determining the coreceptor usage by the infecting viral strain. Bioinformatics programs that predict coreceptor usage could provide an alternative method to screen candidates for treatment with CCR5 antagonists, particularly in countries with limited financial resources. Thus, the present study aims to identify the best approach using bioinformatics tools for determining HIV-1 coreceptor usage in clinical practice. Proviral DNA sequences and Trofile results from 99 HIV-1-infected subjects under clinical monitoring were analyzed in this study. Based on the Trofile results, the viral variants present were 81.1% R5, 21.4% R5X4 and 1.8% X4. Determination of tropism using a Geno2pheno[coreceptor] analysis with a false positive rate of 10% gave the most suitable performance in this sampling: the R5 and X4 strains were found at frequencies of 78.5% and 28.4%, respectively, and there was 78.6% concordance between the phenotypic and genotypic results. Further studies are needed to clarify how genetic diversity amongst virus strains affects bioinformatics-driven approaches for determining tropism. Although this strategy could be useful for screening patients in developing countries, some limitations remain that restrict the wider application of coreceptor usage tests in clinical practice.

A metodologia para amplificação subtipo-específica por PCR da região transmembrana do gene env (gp41) do HIV-1, descrita por Yagyu e colaboradores, foi testada a partir de DNA proviral de 100 pacientes infectados pelo HIV-1 de Itajaí, Sul do Brasil. Setenta indivíduos apresentaram produtos amplificados e correspondentes aos subtipos B, C, D e F de acordo com a metodologia escolhida. Destes indivíduos, 26 (37,1%) apresentaram a amplificação esperada para o subtipo C de acordo com a metodologia; 42 (60%) apresentaram os produtos esperados para os subtipos B e D, sendo que na etapa seguinte de diferenciação destes subtipos, 16 (22,9%) corresponderam ao subtipo D e 26 (37,1%) ao subtipo B. Dois indivíduos (2,9%) mostraram produtos amplificados após a amplificação específica para o subtipo F. O sequenciamento e a comparação com sequências referências confirmou a subtipagem de HIV-1 C e B obtida pela metodologia. No entanto, indivíduos subtipados erroneamente como HIV-1 D e F pela metodologia, foram classificados pela comparação com sequências referências como subtipos C e B, respectivamente. Em relação aos indivíduos que não mostraram produtos amplificados, a baixa carga viral observada no histórico destes pacientes seria em parte responsável pela dificuldade na subtipagem pela metodologia de PCR, como demonstrado pelo resultado significativo no ANOVA ao testar o efeito da carga viral no sucesso da amplificação. O alinhamento das sequências obtidas com sequências referências de HIV-1 correspondentes à região da gp41 demonstrou que há uma alta diversidade intra-subtipo e que as regiões a partir das quais foram desenhados os oligonucleotídeos iniciadores HIV-1 subtipo-específicos não são conservadas nem suficientemente representativas dos subtipos observados nas populações brasileiras para permitir sua correta identificação. Portanto, esta metodologia não é aplicável para populações virais brasileiras.

The method used by YAGYU et al. for the subtype-specific polymerase chain reaction (PCR) amplification of the gp41 transmembrane region of the human immunodeficiency virus type-1 (HIV-1) env gene, was tested. HIV-1 proviral DNA from 100 infected individuals in Itajaí, South Brazil was used to analyze this method. Seventy individuals were determined according to this method as having PCR products at the expected size for subtypes B, C, D and F. Of these individuals, 26 (37.1%) were observed as having the expected amplification for subtype C, and 42 (60%) were observed as having the expected products for subtypes B and D. Of the subtype B and D amplicons, 16 (22.9%) were classified as subtype D, and 26 (37.1%) were classified as subtype B. Two individuals (2.9%) had amplicons that were observed after subtype F-specific amplification was performed. Sequencing and comparing the patient sequences to reference sequences confirmed the classification of sequences of subtypes C and B. However, sequences that were falsely determined as being D and F in the PCR assay were determined as being subtypes C and B, respectively, by sequence analysis. For those individuals from whom no amplified products were obtained, a low viral load that was indicated in their patient history may explain the difficulty in subtyping by PCR methods. This issue was demonstrated by the results of ANOVA when testing the effect of viral load on the success of PCR amplification. The alignment of the obtained sequences with HIV-1 reference sequences demonstrated that there is high intra-subtype diversity. This indicates that the subtype-specific primer binding sites were not conserved or representative of the subtypes that are observed in the Brazilian populations, and that they did not allow the correct classification of HIV-1 subtypes. Therefore, the proposed method by YAGYU et al. is not applicable for the classification of Brazilian HIV-1 subtypes.