Development of a Qualitative Quantitative Polymerase Chain Reaction Test to Identify Patients Failing First-Line Therapy to Non-Nucleotide Reverse Transcriptase Inhibitor.

Antiretroviral therapy (ART) can be compromised by selection of drug resistance strains, which can be promoted by lack of adherence during therapy and drug tolerance, and some of these drug-resistant strains can persist for years as minority populations. The K103N drug resistance mutation is selected by the use of non-nucleotide reverse transcriptase inhibitors, including nevirapine or efavirenz (EFV), used in low-income countries. Here we describe the use of a less expensive qualitative point mutation polymerase chain reaction (PMqPCRK103N) targeting K103N mutation. To validate the use of this methodology, we tested previously sequenced samples from patients treated with highly active ART with viral loads above 2,000 copies/ml and compared the results of our assay with Illumina deep sequencing. Due to its low cost and high specificity, this test is particularly suitable for low-income countries to screen for pretreatment resistance in patients either initiating ART or failing first-line regimens containing EFV.

Clinical and hematological evaluation of Rangelia vitalii-naturally infected dogs in southeastern Brazil

Rangelia vitalii, a tick-borne piroplasm that infects dogs, has been recently molecularly characterized in Brazil, Uruguay and Argentina. Studies on molecular characterization of these piroplasms in different Brazilian regions are scarce. The aim of this study was to evaluate clinical and hematological changes in dogs caused by R. vitalii in the mountainous region of the state of Rio de Janeiro. Blood samples from 36 dogs were evaluated for piroplasms and hematological disorders using light microscopy and molecular analysis. Blood samples from all the animals included in this study were confirmed to be positive for R. vitalii through genetic sequencing. Clinical signspresented by 24 of the 36 dogs of the study were evaluated during appointments or hospitalization within private practice. The most frequent clinical disorders in these dogs that were naturally infected with R. vitalii were fever, spontaneous cutaneous bleeding and diarrhea. Normochromic non-regenerative anemia and thrombocytopenia were the most common hematological disorders in these R. vitalii-positive dogs and therefore should be considered in hematological evaluations on suspected cases.(AU)

Rangelia vitalii, um piroplasma transmitido por carrapatos que infecta cães, foi sendo recentemente caracterizado molecularmente no Brasil, Uruguai e Argentina. Nas diferentes regiões brasileiras são escassos os estudos acerca da caracterização molecular destes piroplasmídeos. O objetivo deste estudo foi avaliar as alterações clínicas e hematológicas em cães causadas por R. vitalii na região serrana do Rio de Janeiro. Amostras de sangue total de 36 cães foram examinadas quanto à presença de piroplasmas pela microscopia de luz, alterações hematológicas e análise molecular. Todos os cães do presente estudo foram positivos para R. vitalii através do sequenciamento genético. Dos 36 animais positivos para R. vitalii, 24 foram avaliados clinicamente. As alterações mais frequentemente observadas foram febre, sangramento cutâneo espontâneo e diarréia. Anemia normocítica normocrômica arregenerativa e trombocitopenia foram as alterações hematológicas mais observadas em cães positivos para R. vitalii, devendo ser consideradas na avaliação hematológica de cães suspeitos.(AU)