Detección de mutaciones del gen 23S de Helicobacter pylori implicadas en la resistencia a claritromicina

Introducción: Uno de los principales factores que influyen en el tratamiento para la erradicación de Helicobacter pylori es la resistencia a antibióticos, la cual difiere entre países e incluso regiones de un país. Entre los antibióticos más usados para el tratamiento de la infección se encuentra la claritromicina, se ha demostrado que el gen 23S ARNr está involucrado en la resistencia a este antibiótico, como resultado de mutaciones puntuales. Objetivo: Detectar las mutaciones presentes en el gen 23S ARNr que codifican la resistencia a la claritromicina en Helicobacter pylori a través de un método no invasivo y rápido. Materiales y métodos: A partir de muestras de heces de 76 pacientes con síntomas gastrointestinales asociados a la bacteria, se aisló y purificó el ADN bacteriano, se identificó el gen 23S ARNr mediante seminested PCR. Para la detección de mutaciones puntuales en el gen se realizó la RFLP, utilizando las enzimas HhaI que detecta la mutación T2717C y MboII que identifica la mutación A2142C/G. Resultados: Un total de 45 pacientes resultaron positivos a Helicobacter pylori lo cual corresponde al 59,2%. La mutación T2717C analizada con la enzima HhaI se presentó en el 2,2% de la muestra de estudio, no se obtuvo resultados positivos para la enzima MboII. Conclusiones: A través de la Seminested PCR se identificó al gen 23S ARNr de Helicobacter pylori, PCR-RFLP es un método fiable para detectar la presencia de mutaciones causantes de resistencias a antibióticos, útil antes de elegir el tratamiento erradicador contra las infecciones por Helicobacter pylori.

Investigasi resistensi Anopheles sp. terhadap insektisida piretroid dan kemungkinan terjadinya mutasi gen voltage gated sodium channel (VGSC)

Penggunaan insektisida golongan piretroid secara luas dan terus menerus dalam pencegahan penularan malaria dapat menimbulkan mutasi gen voltage gate sodium channel (VGSC) pada vektor nyamuk yang dikenal dengan mutasi knockdown resistance (kdr). Mutasi gen ini membuat sensitifitas ikatan protein VGSC dengan insektisida menurun sehingga menyebabkan resistensi. Timbulnya resistensi insektisida piretroid pada nyamuk vektor dapat menjadi penghambat keberhasilan pemutusan penularan penyakit malaria sehingga deteksi adanya mutasi mutlak diperlukan. Tujuan penelitian ini untuk mengetahui status resistensi insektisida piretroid dan mengidentifikasi mutasi pada gen VGSC kodon 1014 penanda resistensi nyamuk Anopheles sp. yang menjadi vektor malaria di Provinsi Sumatera Selatan. Sampel diambil di tiga Kabupaten, yaitu Muara Enim, Ogan Komering Ulu (OKU), dan Lahat. Uji awal kerentanan terhadap insektisida piretroid (permetrin 0,75%) dilakukan sesuai standar WHO tahun 2016 pada spesies dominan, yaitu Anopheles vagus Dönitz. Selanjutnya, identifikasi mutasi gen VGSC pada kodon 1014 dilakukan pada An. vagus dan spesies Anopheles barbirostris van der Wulp yang tertangkap, dengan teknik seminested-PCR dan dilanjutkan dengan analisis sekuensing. Hasil uji kerentanan Anopheles sp. di Kabupaten Muara Enim menunjukkan resisten terhadap piretroid, sedangkan di Kabupaten Lahat dan OKU masih rentan terhadap insektisida piretroid. Sementara, analisis sekuensing PCR menunjukkan tidak terjadi perubahan basa alel kdr pada target site insektisida gen VGSC sehingga dapat disimpulkan secara molekuler belum ditemukan adanya mutasi pada gen VGSC kdr allel penanda resistensi insektisida piretroid.

Nonpolio enteroviruses can also be recovered from non-reproducible cytopathology in L20B cell line

ABSTRACTSamples showing cytopathology (CPE) on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in L20B or RD are considered negative for both poliovirus and nonpolio enteroviruses (NPEVs). The phenomenon is termed ‘non-reproducible CPE’. Its occurrence is usually ascribed to the likely presence of reoviruses, adenoviruses and other non-enteroviruses. This study aimed to investigate the likelihood that NPEVs are also present in cases with non-reproducible CPE.Twenty-six (26) cell culture suspensions were analyzed in this study. The suspensions were collected from the WHO National Polio Laboratory, Department of Virology, College of Medicine, University of Ibadan. The suspensions emanated from 13 L20B cell culture tubes that showed cytopathology within 5 days of inoculation with fecal suspension from AFP cases. However, on passage into one each of RD and L20B cell lines, the CPE was not reproducible. All samples were subjected to RNA extraction, cDNA synthesis, the WHO recommended VP1 RT-seminestedPCR assay, species resolution PCR assay, sequencing and phylogenetic analysis.Six (6) samples were positive for the VP1 RT-seminested PCR assay. Only four of which were positive by the species resolution PCR assay. The four amplicons were sequenced, however, only three (3) were successfully identified as Coxsackievirus A20 (2 isolates) and Echovirus 29 (1 isolate).The results of this study unambiguously showed the presence of NPEVs (particularly CVA20 and E29) in cell culture supernatants of samples with CPE on initial inoculation into L20B cell line but with no observed or reproducible CPE on passage in RD cell line. Therefore, like reoviruses, adenoviruses and other non-enteroviruses, NPEVs can also be recovered in cases with non-reproducible CPE.

Development of Enhanced Primer Sets for Detection of Norovirus

Norovirus (NV) is a major viral pathogen that causes nonbacterial acute gastroenteritis and outbreaks of food-borne disease. The genotype of NV most frequently responsible for NV outbreaks is GII.4, which accounts for 60–80% of cases. Moreover, original and new NV variant types have been continuously emerging, and their emergence is related to the recent global increase in NV infection. In this study, we developed advanced primer sets (NKI-F/R/F2, NKII-F/R/R2) for the detection of NV, including the variant types. The new primer sets were compared with conventional primer sets (GI-F1/R1/F2, SRI-1/2/3, GII-F1/R1/F2, and SRII-1/2/3) to evaluate their efficiency when using clinical and environmental samples. Using reverse transcription polymerase chain reaction (RT-PCR) and seminested PCR, NV GI and GII were detected in 91.7% (NKI-F/R/F2), 89.3% (NKII-F/R/R2), 54.2% (GI-F1/R1/F2), 52.5% (GII-F1/R1/F2), 25.0% (SRI-1/2/3), and 32.2% (SRII-1/2/3) of clinical and environmental specimens. Therefore, our primer sets perform better than conventional primer sets in the detection of emerged types of NV and could be used in the future for epidemiological diagnosis of infection with the virus.

Clinical, Hematologic, and Molecular Findings in Naturally OccurringBabesia canis vogeliin Egyptian Dogs

Background. Canine babesiosis is a clinically important hemoprotozoan parasite affecting dogs. The goal of this present study was to determine the clinical symptoms and to establish its hematological and microscopic detection and compare it with the PCR findings attained from dogs infected withBabesia canis vogeli.Methodology/Principal Findings. 13-PCR confirmed Babesia-infected dogs were examined; seminested PCR was used to discover the precise type ofBabesiaandBabesia canis vogeliwas the only subspecies detected. The most consistent clinical signs were elevated rectal temperature and a pale mucous membrane. Thrombocytopenia, monocytosis, and lymphocytosis, along with a significant reduction in red cell parameters, were the most commonly recorded hematologic alterations. Microscopic examination revealed the presence of typical large merozoites and trophozoites ofB. canisin the ratio 76.92%.Conclusions/Significance. The presumptive diagnosis of canine babesiosis should be based on a fever and anemia, while thrombocytopenia is considered the hallmark of the disease; microscopic examination may not be very revealing in the detection at low parasitemia, but it remains the most rapid confirmatory method. Seminested PCR turned out to be a sensitive and accurate method for diagnosis; during the process of differentiation betweenBabesiasubspecies, onlyB. canis subsp. vogeliwas detected.