ABSTRACT Background: Zika virus (ZIKV) sexual transmission and prolonged viral shedding in semen have been previously reported, suggesting a strong viral affinity for genital tissues. A transient impact of ZIKV on male fertility was shown in animal and human studies. Methods: Adult male patients with confirmed ZIKV infection diagnosed in the city of Araraquara, Brazil during the epidemic season of 2016 were invited one year after the acute infection to respond to a questionnaire of genital symptoms and to provide a semen sample for molecular ZIKV testing and spermogram analysis, as well as a serum sample for hormonal testing. Results: 101 of 187 tested patients had positive ZIKV RT-PCR in plasma and/or urine samples (54%, 72 women and 29 men). Of 15 adult male participants for whom telephone contact was successful, 14 responded to the questionnaire of genital symptoms and six consented to provide a semen sample at a median of 12 months after the acute infection. We report abnormal spermogram results from patients one year after confirmed ZIKV infection. Conclusions: Our findings suggest a possible long-term detrimental effect of ZIKV infection on human male fertility that has to be further explored in well-characterized samples from cohort studies conducted in ZIKV-endemic areas.

HHV-6 é o agente etiológico do Exantema Súbito e considerado a sexta doença mais comum na infância. Em indivíduos imunocomprometidos, a reativação da infecção latente pode causar doença aguda ou morte. Padronizamos PCR em Tempo Real utilizando a química Sybr Green na detecção do HHV-6 e comparamos os resultados com a PCR convencional. Um fragmento de 214 pb foi clonado através do kit pGEM-T do sistema Promega. Com este clone, foram feitas diluições seriadas em um pool de leucócitos negativos a partir de 10-6 ng/µL (equivalente a 2465,8 moleculas/µL) até 10-9 (equivalente a 2,46 moleculas/µL). As diluições foram amplificadas por PCR em Tempo Real utilizando Sybr Green, com primers HHV3 5' TTG TGC GGG TCC GTT CCC ATC ATA 3' e HHV4 5' TCG GGA TAG AAA AAC CTA ATC CCT 3' e pelo método convencional, PCR nested usando primers HHV1 (externo): 5' CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (externo): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3', HHV3 (interno) e HHV4 (interno): 5' TCG GGA TAG AAA AAC CTA ATC CCT 3'. O limite de detecção foi determinado pelas diluições seriadas do plasmídio contendo um fragmento de HHV6: para o ensaio com Sybr Green, foi de 24,6 moleculas/µL e para a PCR nested, 2,46 moleculas/µL. Elegemos o PCR em Tempo Real - Sybr Green como método diagnóstico e quantitativo do HHV-6 devido a sua boa sensibilidade e menor risco de contaminação.

HHV-6 is the etiological agent of Exanthem subitum which is considered the sixth most frequent disease in infancy. In immuno-compromised hosts, reactivation of latent HHV-6 infection may cause severe acute disease. We developed a Sybr Green Real Time PCR for HHV-6 and compared the results with nested conventional PCR. A 214 pb PCR derived fragment was cloned using pGEM-T easy from Promega system. Subsequently, serial dilutions were made in a pool of negative leucocytes from 10-6 ng/µL (equivalent to 2465.8 molecules/µL) to 10-9 (equivalent to 2.46 molecules/µL). Dilutions of the plasmid were amplified by Sybr Green Real Time PCR, using primers HHV3 (5' TTG TGC GGG TCC GTT CCC ATC ATA 3)'and HHV4 (5' TCG GGA TAG AAA AAC CTA ATC CCT 3') and by conventional nested PCR using primers HHV1 (outer): 5'CAA TGC TTT TCT AGC CGC CTC TTC 3'; HHV2 (outer): 5' ACA TCT ATA ATT TTA GAC GAT CCC 3'; HHV3 (inner) and HHV4 (inner) 3'. The detection threshold was determined by plasmid serial dilutions. Threshold for Sybr Green real time PCR was 24.6 molecules/µL and for the nested PCR was 2.46 molecules/µL. We chose the Real Time PCR for diagnosing and quantifying HHV-6 DNA from samples using the new Sybr Green chemistry due to its sensitivity and lower risk of contamination.

O aumento da sobrevida dos pacientes que utilizam terapêutica antiretroviral altamente eficaz (HAART- Highly Active Antiretroviral Therapy) trouxe uma nova demanda de casais sorodiscordantes que desejam filhos. Como esses casais não podem abandonar o uso de preservativos, torna-se indispensável tratar o sêmen infectado com técnicas laboratoriais eficazes que além de isolar os melhores espermatozóides, reduzam a carga viral do HIV e HCV a níveis indetectáveis. Para isso, são utilizadas técnicas de semen washing, associadas a testes ultra sensíveis de biologia molecular. Após análise seminal, sêmen de 20 pacientes co-infectados HIV-HCV foram submetidos a fracionamento celular e isolamento de espermatozóides móveis através de método de densidade de gradiente descontínuo e swim-up. Posteriormente, testes para detecção do RNA do HIV e HCV foram aplicados nos sêmens totais e frações seminais obtidas. Em fase pré semen washing, o HIV foi detectado em 100% dos semens totais. Contrariamente, o HCV foi detectado em apenas uma amostra. Em fase pós semen washing, o HIV e HCV não foram detectados em nenhuma das frações seminais. A redução do HIV e do HCV através de semen washing mostra-se um método eficaz a indivíduos co-infectados HIV-HCV, apesar do encontro do HCV no sêmen ser raro.

INTRODUCTION: Prolonged survival of patients under HAART has resulted in new demands for assisted reproductive technologies. HIV serodiscordant couples wish to make use of assisted reproduction techniques in order to avoid viral transmission to the partner or to the newborn. It is therefore essential to test the effectiveness of techniques aimed at reducing HIV and HCV loads in infected semen using molecular biology tests. METHODS: After seminal analysis, semen samples from 20 coinfected patients were submitted to cell fractioning and isolation of motile spermatozoa by density gradient centrifugation and swim-up. HIV and HCV RNA detection tests were performed with RNA obtained from sperm, seminal plasma and total semen. RESULTS: In pre-washing semen, HIV RNA was detected in 100% of total semen samples, whereas HCV RNA was concomitantly amplified in only one specimen. Neither HIV nor HCV were detected either in the swim-up or in the post-washing semen fractions. CONCLUSIONS: Reduction of HIV and/or HCV shedding in semen by density gradient centrifugation followed by swim-up is an efficient method. These findings lead us to believe that, although semen is rarely found to contain HCV, semen processing is highly beneficial for HIV/HCV coinfected individuals.