Impact of time of culture specimen collection on the recovery of Neisseria gonorrhoeae after a positive nucleic acid amplification test.

OBJECTIVES: Culture of Neisseria gonorrhoeae remains essential for antimicrobial resistance (AMR) surveillance. We evaluated the effect of time of specimen collection on culture yield following a positive nucleic acid amplification test (NAAT). METHODS: We retrospectively assessed N. gonorrhoeae culture yield among asymptomatic individuals (largely men who have sex with men) who attended for sexual health screening and had a positive NAAT. Participants underwent either same-day testing and notification (Drassanes Exprés) or standard screening with deferred testing. RESULTS: Among 10 423 screened individuals, 809 (7.7%) tested positive for N. gonorrhoeae. A total of 995 different anatomical sites of infection culture was performed in 583 of 995 (58.6%) of anatomical sites (Drassanes Exprés 278 of 347, 80.1%; standard screening 305 of 648, 47.1%; p<0.001). Recovery was highest when culture specimens were collected within 3-7 days of screening with only a slight drop in recovery when the interval extended to 7 days . Recovery from pharynx was 38 of 149 (25.5%) within 3 days, 19 of 81 (23.4%) after 4-7 days (p=0.7245), 11 of 102 (10.7%) after 8-14 days (p<0.0036) and 1 of 22 (4.5%) with longer delays (p=0.00287). Recovery from rectum was 49 of 75 (65.3%) within 3 days, 28 of 45 (62.2%) after 4-7 days (p=0.7318), 41 of 69 (59.4%) after 8-14 days (p=0.4651) and 6 of 18 (33.3%) with longer delays (p=0.0131). Median culture specimen collection time was 1 day within Drassanes Exprés vs 8 days within standard screening. Consequently, the overall culture yield was slightly higher within Drassanes Exprés (102/278, 36.6% vs 99/305, 32.5%; p=0.2934). CONCLUSION: Reducing the interval between screening and collection of culture specimens increased N. gonorrhoeae recovery in extragenital samples. Implementing a same-day testing and notification programme increased collection of culture samples and culture yield in our setting, which may help AMR surveillance.

Cross-sectional evaluation of circulating hepatitis B virus RNA and DNA: Different quasispecies?

BACKGROUND: Different forms of pregenomic and other hepatitis B virus (HBV) RNA have been detected in patients' sera. These circulating HBV-RNAs may be useful for monitoring covalently closed circular DNA activity, and predicting hepatitis B e-antigen seroconversion or viral rebound after nucleos(t)ide analog cessation. Data on serum HBV-RNA quasispecies, however, is scarce. It is therefore important to develop methodologies to thoroughly analyze this quasispecies, ensuring the elimination of any residual HBV-DNA. Studying circulating HBV-RNA quasispecies may facilitate achieving functional cure of HBV infection. AIM: To establish a next-generation sequencing (NGS) methodology for analyzing serum HBV-RNA and comparing it with DNA quasispecies. METHODS: Thirteen untreated chronic hepatitis B patients, showing different HBV-genotypes and degrees of severity of liver disease were enrolled in the study and a serum sample with HBV-DNA > 5 Log10 IU/mL and HBV-RNA > 4 Log10 copies/mL was taken from each patient. HBV-RNA was treated with DNAse I to remove any residual DNA, and the region between nucleotides (nt) 1255-1611 was amplified using a 3-nested polymerase chain reaction protocol, and analyzed with NGS. Variability/conservation and complexity was compared between HBV-DNA and RNA quasispecies. RESULTS: No HBV-DNA contamination was detected in cDNA samples from HBV-RNA quasispecies. HBV quasispecies complexity showed heterogeneous behavior among patients. The Rare Haplotype Load at 1% was greater in DNA than in RNA quasispecies, with no statistically significant differences (P = 0.1641). Regarding conservation, information content was equal in RNA and DNA quasispecies in most nt positions [218/357 (61.06%)]. In 102 of the remaining 139 (73.38%), HBV-RNA showed slightly higher variability. Sliding window analysis identified 4 hyper-conserved sequence fragments in each quasispecies, 3 of them coincided between the 2 quasispecies: nts 1258-1286, 1545-1573 and 1575-1604. The 2 hyper-variable sequence fragments also coincided: nts 1311-1344 and 1461-1485. Sequences between nts 1519-1543 and 1559-1587 were only hyper-conserved in HBV-DNA and RNA, respectively. CONCLUSION: Our methodology allowed analyzing HBV-RNA quasispecies complexity and conservation without interference from HBV-DNA. Thanks to this, we have been able to compare both quasispecies in the present study.

The high genetic similarity between rhinoviruses and enteroviruses remains as a pitfall for molecular diagnostic tools: A three-year overview.

BACKGROUND: Enteroviruses (EVs) and rhinoviruses (RVs) belong to the Enterovirus genus within the Picornaviridae family, and show genetic similarities. These viruses are related to mild diseases, but EVs infections can sometimes lead to more severe complications. Current diagnostic molecular techniques should discriminate between the four EV and the three RV species that infect humans. The aim was to revise the EV and RV PCR-confirmed specimens by sequencing for genetic characterisation. MATERIAL AND METHODS: Respiratory tract specimens were collected from patients with suspicion of respiratory infection. Respiratory viruses' laboratory-confirmation was performed by commercial multiplex real-time RT-PCR assays. Genetic characterisation of all EV and in a selection of RV was performed based on the phylogenetic analyses of partial VP1 and VP4/2 sequences, respectively. RESULTS: From 19,957 tested specimens, 309 (1.5%) were EV-positive, 2546 (12%) were RV-positive, and 233 (1%) were EV/RV co-detections. The phylogenetic analyses revealed that: among single EV detections, 177/309 (57%) were characterised as EV, 2/309 (1%) as RV, and 130/309 (42%) could not be typed; among single 1771 RV detections (Ct < 35), 1651/1771 (93%) were characterised as RV, 3/1771 (0.3%) as EV and 117/1771 (6.7%) could not be typed. Among EV/RV co-detections, 62/233 (27%) were characterised as EV, 130/233 (56%) as RV and 41/233 (18%) could not be typed. CONCLUSIONS: A diagnostic method well considered for routine laboratory-confirmation of respiratory viruses should discriminate EV and RV targets. RVs are usually associated with mild respiratory disease, but the potential relatedness of EVs to neurological complications makes their monitoring mandatory. Therefore, an accurate detection and differentiation should be required in commercial diagnostic solutions.