Parechovirus epidemiology and disease association are not fully understood. Real-time reverse transcriptase PCR (RT-PCR) for all human parechoviruses (HPeV) was applied on stool samples from two groups of patients. Both groups contained patients with acute enteritis of all age groups, seen during one full year. Patients with norovirus, adenovirus, enterovirus, astrovirus, or rotavirus infections were excluded. In 118 patients from outbreak and hospital settings, no HPeV was detected. In a prospective study group of 499 nonhospitalized patients, the detection rate was 1.6%. One virus-positive patient was detected from 39 control patients. Positive samples occurred only in summer and autumn. Only one patient had accompanying respiratory symptoms. An association with travel or animal contact was not found. All positive patients except one were <2 years of age, with a neutral gender ratio. In children <2 years of age, the detection rate was 11.6% (7 of 60 children). The range of viral loads was 3,170 to 503,377,290 copies per gram or milliliter of stool. One of the highest viral loads occurred in a control child without symptoms at the time of testing. Phylogenetic analysis showed mainly contemporary HPeV1 strains in our patients but also showed a separate new lineage of HPeV1 in evolutionary transition from the historical prototype strain. Moreover, a novel sixth HPeV type was identified. Full genome analysis of the two viruses revealed recombination between HPeV1 and -3 in one and HPeV6 and -1 in another. HPeV seems relevant in children <2 years and specific RT-PCR for HPeV should be included in enteritis screening.

Cardioviruses cause serious disease, mainly in rodents, including diabetes, myocarditis, encephalomyelitis, and multiple sclerosis-like disseminated encephalomyelitis. Recently, a human virus isolate obtained 25 years ago, termed Saffold virus, was sequenced and classifi ed as a cardiovirus. We conducted systematic molecular screening for Saffold-like viruses in 844 fecal samples from patients with gastroenteritis from Germany and Brazil, across all age groups. Six cardioviruses were identifi ed in patients <6 years of age. Viral loads were 283,305-5,044,412,175 copies/g of stool. Co-infections occurred in 4 of 6 children. No evidence for outbreak-like epidemic patterns was found. Phylogenetic analysis identifi ed 3 distinct genetic lineages. Viral protein 1 amino acids were 67.9%-77.7% identical and had a distance of at least 39.4% from known cardioviruses. Because closely related strains were found on 2 continents, global distribution in humans is suspected. Saffold-like viruses may be the fi rst human cardiovirus species to be identifi ed.

A nonfluorescent low-cost, low-density oligonucleotide array was designed for detecting the whole coronavirus genus after reverse transcription (RT)-PCR. The limit of detection was 15.7 copies/reaction. The clinical detection limit in patients with severe acute respiratory syndrome was 100 copies/sample. In 39 children suffering from coronavirus 229E, NL63, OC43, or HKU1, the sensitivity was equal to that of individual real-time RT-PCRs.Coronaviruses (CoV) (family Coronaviridae, order Nidovirales) are large enveloped RNA viruses with a 27-to 32-kb genome of positive polarity. They comprise a very diverse spectrum of pathogens of humans and animals (2, 7). The coronavirus etiology of severe acute respiratory syndrome (SARS) and the recent discoveries of the novel human coronaviruses (hCoV) NL63 and HKU1 (5, 13, 15) have triggered intensified efforts in virus identification and diagnostics. Generic reverse transcription (RT)-PCR assays with a very broad detection range are required, but few such assays are available. None of them has been previously validated in a diagnostic setting (9, 12).The requirement for sequencing in order to achieve strain identification limits the applicability of generic PCR assays in general. Alternative techniques, such as mass spectrometry or complex fluorescent DNA microarrays, have been proposed (10), but these will often be too sophisticated for medical facilities. We describe here a simple and feasible approach to detecting the full spectrum of coronaviruses with diagnostic sensitivity, combining generic RT-PCR and low-cost, low-density (LCD) DNA microarrays which can be read with the naked eye.Primers for universal RT-PCR for the genus Coronavirus were designed after aligning all coronavirus RNA-dependent RNA polymerase genes. RNA-dependent RNA polymerase motifs A and C were targeted because they contain short amino acid patterns that are 100% identical in all coronaviruses (16). Primer binding regions corresponded to patterns LMGWDYPKCD and MMILSDDAV, comprising domains essential for metal ion chelation and binding of the primer 3Ј-end/template complex (11, 16). Reactions (25-l mixtures) were carried out using the QIAGEN (Hilden, Germany) one-step RT-PCR kit, with 200 nM of primer PC2S2 (equimolar mixture of TTATGGGTTGGGAT TATC and TGATGGGATGGGACTATC), 900 nM of primer PC2As1 (equimolar mixture of TCATCACTCAGAATCATCA, TCATCAGAAAGAATCATCA, and TCGTCGGACAAGATC ATCA), 1 l QIAGEN one-step RT-PCR kit enzyme mix, and 5 l RNA extract. The amplification procedure comprised 30 min at 50°C; 15 min at 95°C; 10 cycles of 20 s at 94°C, 30 s starting at 62°C with a decrease of 1°C per cycle, and 40 s at 72°C; and 30 cycles of 20 s at 95°C, 30 s at 52°C, and 40 s at 72°C. To determine the sensitivity of the assay, the target regions including sufficient stretches of flanking sequence were cloned from several coronaviruses (Table 1) and transcribed into RNA (3, 4). Amplification

Background: Current HIV-1 viral-load assays are too expensive for resource-limited settings. In some countries, monitoring of antiretroviral therapy is now more expensive than treatment itself. In addition, some commercial assays have shown shortcomings in quantifying rare genotypes. Methods: We evaluated real-time reverse transcription-PCR with internal control targeting the conserved long terminal repeat (LTR) domain of HIV-1 on reference panels and patient samples from Brazil (n ‫؍‬ 1186), South Africa (n ‫؍‬ 130), India (n ‫؍‬ 44), and Germany (n ‫؍‬ 127). Results: The detection limit was 31.9 IU of HIV-1 RNA/mL of plasma (>95% probability of detection, Probit analysis). The internal control showed inhibition in 3.7% of samples (95% confidence interval, 2.32%-5.9%; n ‫؍‬ 454; 40 different runs). Comparative qualitative testing yielded the following: Roche Amplicor vs LTR assay (n ‫؍‬ 431 samples), 51.7% vs 65% positives;

SARS coronavirus-2 (SARS-CoV-2) detection in different clinical specimens has raised important insights about its pathogenesis, but some details remain to be understood. In that respect, disrupt viral control seen in solid organ transplant patients on chronic immunosuppression can help unveil pathogenic mechanisms and characterize new coronavirus disease-19 (COVID-19) immunological and clinical aspects, as well as secondary complications. We herein report a case of SARS-CoV-2 detection in ascitic fluid from a kidney transplant patient with decompensated cirrhosis and COVID-19 and then discuss about immune, cellular, and virological aspects of such clinical presentation of the disease, which also included a disseminated infection, demonstrated by viral detection in his blood sample. We subsequently discuss about the fatal outcome caused by a secondary bloodstream infection by Cryptococcus neoformans. This unprecedented case report presents ascitic fluid as a novel specimen in which SARS-CoV-2 can be detected. Immune dysregulation and cumulative risk factors may lead to secondary infections by opportunistic agents, including Cryptococcus neoformans.