SARS-CoV-2 viral load dynamics and real-time RT-PCR cycle threshold interpretation in symptomatic non-hospitalised individuals in New Zealand: a multicentre cross sectional observational study.

We conducted a multicentre cross sectional observational study of laboratory, public health and hospitalisation data for PCR-confirmed COVID-19 cases within the New Zealand Northern Region, between 12 February and 8 June 2020. The aim of this study was to describe population level SARS-CoV-2 upper respiratory tract (URT) viral load dynamics by stratifying positivity rates and polymerase chain reaction (PCR) cycle threshold (Ct) values of URT samples from COVID-19 cases by days since symptom onset, and to explore utility of Ct values in determining length of time post-infection and thus potential infectivity. Of 123,124 samples tested for SARS-CoV-2 by PCR, 579 samples (407 positive and 172 negative) from 368 symptomatic non-hospitalised individuals with PCR-confirmed infection were included. Sample positivity rate was 61.5% (8/13) for pre-symptomatic samples, rising to 93.2% (317/340) for samples collected during the purported symptomatic infectious period (days 0-10 post-symptom onset), and dropping to 36.3% (82/226) for post-infectious period samples (day 11 onwards). URT viral load peaked shortly after symptom onset, with median Ct values ranging 20.00-29.99 until 15 days post-symptom onset, and >30.00 after this time. Of samples with a Ct value of <20.00, 96.1% were collected during the symptomatic infectious period. However, of samples with a Ct value ≥30.00 and ≥35.00, 46.9% and 18.5%, respectively, were also collected during the symptomatic infectious period. The findings of this study indicate that at or soon after symptom onset represents the optimum time to test for SARS-CoV-2 in the URT, with median Ct values suggesting the useful testing window extends until around 15 days post-symptom onset. In asymptomatic individuals or those with unknown dates of symptom onset, Ct values <20.00 imply recent onset/potential infectivity, but Ct values ≥30.00 or ≥35.00 do not exclude recent onset/potential infectivity. Individual sample Ct values should not be used as an absolute marker of length of time post-infection or to exclude infectivity where date of symptom onset is unavailable.

Impact of the COVID-19 nonpharmaceutical interventions on influenza and other respiratory viral infections in New Zealand.

Stringent nonpharmaceutical interventions (NPIs) such as lockdowns and border closures are not currently recommended for pandemic influenza control. New Zealand used these NPIs to eliminate coronavirus disease 2019 during its first wave. Using multiple surveillance systems, we observed a parallel and unprecedented reduction of influenza and other respiratory viral infections in 2020. This finding supports the use of these NPIs for controlling pandemic influenza and other severe respiratory viral threats.

An understanding of discordant SARS-CoV-2 test results: an examination of the data from a central Auckland laboratory.

AIM: The diagnostic sensitivity of the SARS-CoV-2 real time reverse transcription polymerase chain reaction (RT-PCR) test has not been determined. This has led to a degree of uncertainty in the interpretation of results, particularly in patients tested repeatedly. The aim of this study was to explore the characteristics of patients who initially tested negative, and subsequently tested positive for SARS-CoV-2. METHODS: This retrospective observational study utilised data from the LabPlus Virology laboratory, Auckland City Hospital, to identify cases (hospital and community) with initial negative and subsequent positive SARS-CoV-2 RT-PCR results. Their clinical and laboratory characteristics were summarised. RESULTS: From 1 February to 13 April a total of 20,089 samples were received for SARS-CoV-2 testing. Of 2,011 samples from patients with multiple tests, 25 samples were positive. Nine samples were from patients who initially tested negative then tested positive. Reasons for the initial negative test results, which were all from upper respiratory tract samples, included pre-symptomatic presentation or late presentation. All patients had significant risk factors and ongoing or evolving symptoms, which warranted repeat testing. CONCLUSION: Few patients had discordant test results for SARS-CoV-2 RT-PCR. For patients who have a significant risk factor and a negative test result, repeat testing should be performed.

An observational study of high- and low-abundance anti-retroviral resistance mutations among treatment-naïve people living with HIV in New Zealand between 2012 and 2017.

HIV resistance genotyping detects drug resistance mutations (DRMs) in ≥20% of circulating virus within an infected individual (high-abundance DRMs). Deep sequencing also detects DRMs in smaller viral subpopulations (low-abundance DRMs), although these are of uncertain importance. In this retrospective analysis of 292 treatment-naïve patients, high-abundance DRMs were present in 30/292 (10%) patients, but only one (0.3%) had resistance to first-line anti-retrovirals. Low-abundance DRMs were present in 36/247 (15%) patients, but none who received anti-retrovirals for which these were present had virologic failure. These findings demonstrate that starting first-line therapy in treatment-naïve patients need not be delayed while awaiting resistance testing.

Human papillomavirus and oropharyngeal squamous cell carcinoma: a New Zealand cohort study.

BACKGROUND: Human papillomavirus (HPV)-related oropharyngeal squamous cell carcinomas (OPSCC) are clinically, epidemiologically and prognostically distinct from other OPSCCs. The incidence of HPV-related OPSCCs has increased significantly worldwide over the past few decades. However, no studies of OPSCC with direct molecular HPV testing has been conducted in New Zealand. AIMS: To estimate the proportion of OPSCCs attributable to HPV infections in a New Zealand population with a validated HPV testing algorithm. METHODS: HPV-status was determined by p16 immunohistochemistry and polymerase chain reaction (PCR) of both L1 and E6/7 genes on 55 OPSCCs diagnosed in 2010 and 2011 in Central and South Auckland. Baseline and survival analyses were performed according to HPV status. RESULTS: Forty-one (75%) of OPSCC tumours had HPV infections. There was 98% concordance between p16 immunohistochemistry and real-time E6/E7 PCR. After a median follow-up period of 2.6 years, patients with OPSCC of HPV aetiology had more favourable outcomes compared to patients with HPV-negative OPSCC (hazard ratio 0.14, P = 0.02) after adjustment for other variables. CONCLUSION: This study highlights the significant role that HPV plays in the aetiology of OPSCC in New Zealand, and confirms the high rate of accuracy of p16 immunostaining.

Distribution of HPV genotypes in women with cervical cancer in Auckland, New Zealand; a review of 50 specimens between 2000-2006.

BACKGROUND: In New Zealand, around two hundred women are diagnosed with cervical cancer annually, with approximately seventy deaths from cervical cancer per year. AIM: Our aim was to determine the distribution of oncogenic HPV genotypes in biopsy specimens from women with diagnosed cervical cancers in the Auckland region of New Zealand between 2000-2006. MATERIALS AND METHODS: Confirmed cases of cervical carcinoma were identified from the local pathology register, and representative tissue samples were taken from these blocks. Sections were deparaffinised, and DNA was extracted according to standard protocols. Samples were subject to PCR amplification using L1 consensus primer sets MY09/11 and GP5/6. Further type-specific amplification was performed on positive samples, using an in-house primer sequence based on target sequences within the E6 gene. Remaining samples were typed by a Linear Array Assay, or by DNA sequencing. RESULTS: HPV DNA was detected in 100% of cases. In 49/50 samples, the HPV genotype was identified, with a total of 14 different HPV genotypes detectable. Together HPV-16 and 18 were found in 41/49 cases (83.6%) either singly or in combination. DISCUSSION: Our findings suggest that the distribution of HPV genotypes in New Zealand is similar to that of other geographic areas. CONCLUSION: Ongoing surveillance is warranted to ensure appropriate genotype selection for prophylactic HPV vaccinations.

The effect of DNA methylation inhibitor 5-Aza-2'-deoxycytidine on human endometrial stromal cells.

BACKGROUND: Decidualization, the differentiation of endometrial stromal cells is a crucial step for successful implantation of an embryo, development of the placenta and completion of pregnancy to term. Epigenetic mechanisms are thought to be strongly involved in the regulation of processes controlling implantation, placentation, organ formation and foetal growth. Recent studies suggest that decreased DNA methylation facilitates a receptive endometrium. Hence, the aim of this project was to compare the transcriptional profile changes induced by the inhibitor of DNA methylation, 5-Aza-2'-deoxycytidine (AZA) to the transcriptional changes that happen during decidualization. METHODS AND RESULTS: When DNA methylation was inhibited in a human endometrial stromal cell (HESC) line with AZA, it resulted in the fibroblast-like stromal cells being transformed into decidual-like morphology after 9 days. Expression of both prolactin and insulin-like growth factor binding protein-1, the two established decidualization marker genes, were minimally up-regulated by AZA after 10 days of treatment. In a microarray of a three-way experiment between AZA-treated and oestradiol/progestin/cAMP-treated [medroxy-progesterone acetate (MPA)-mix] HESC and the untreated controls, we detected more than 1000 common genes that had a significant difference of expression compared with the controls. AZA-treated cells in the microarray significantly expressed 76 genes in common with the MPA-mix treated cells, and AZA treatment also differentially regulated 148 genes independently to that of MPA-mix treatment. The MPA-mix regulated at least 36 genes in the cell adhesion, extracellular matrix remodelling and RhoGTPase cytoskeletal reorganization pathway; AZA regulated 19 of these genes in common and 15 other RhoGTPase pathway genes. CONCLUSION: AZA induced some decidualization-like responses of endometrial stromal cells independently of progestins or cAMP, possibly via the cytoskeletal reorganization pathway of the RhoGTPase family.

Epigenetic regulation of E-cadherin controls endometrial receptivity.

Key to the success of human reproduction is the capacity of an embryo to attach and implant into the endometrial wall after which a nutrient supply is established through placentation. Herein, we have examined the potential epigenetic regulation of uterine receptivity by use of the receptive RL95-2 and nonreceptive AN3-CA endometrial epithelial carcinoma cell lines. Using an in vitro model of embryo implantation, we demonstrate that inhibition of DNA methylation by 5'-aza-2'-deoxycytidine (AZA), resulted in the nonreceptive AN3-CA cell line becoming receptive to BeWo cell spheroid attachment. Examination of components of the adherens junction complex revealed that AZA specifically increased the expression of E-cadherin and plakoglobin at the mRNA and protein levels in AN3-CA cells, and E-cadherin protein expression was found to localize to sites of intercellular contact. Forced expression of E-cadherin in AN3-CA cells significantly enhanced receptivity. Small interfering RNA (siRNA)-mediated depletion of the individual DNA methyltransferase (DNMT) molecules did not induce E-cadherin expression in AN3-CA cells; however, concomitant siRNA-mediated depletion of both DNMT3A and DNMT3B induced the expression of E-cadherin. Furthermore, E-cadherin expression was significantly increased after the concomitant siRNA-mediated depletion of DNMT-1, -3A, and -3B in AN3-CA cells. Therefore, we have provided evidence that E-cadherin plays an important role in uterine receptivity and that E-cadherin expression is epigenetically regulated in AN3-CA cells, suppressed by the combined actions of DNMT-1, -3A, and -3B.

Epigenetic regulation of human trophoblastic cell migration and invasion.

Pivotal to successful mammalian reproduction is the ability of a developing embryo to implant to the uterine wall and establish a nutrient supply via placentation. Herein, we have examined the potential epigenetic regulation of human trophoblastic cell migration and invasion by use of the choriocarcinoma cell line, BeWo. Treatment of BeWo cells with a DNA methyltransferase inhibitor, 5'-aza-2'-deoxycytidine (AZA), resulted in conversion of cell morphology to a nonmigratory phenotype. This was exemplified by the ability of AZA to prevent BeWo cell migration in wound healing and transwell migration assays. AZA consequently inhibited BeWo cell invasion through reconstituted basement membrane. Examination of components of the adherens junction complex pivotal for determination of cell phenotype revealed that AZA specifically increased the mRNA level of E-cadherin and plakoglobin (gamma-catenin), but not alpha-catenin and beta-catenin. AZA also increased the gene promoter activity of both plakoglobin and E-cadherin. Protein levels of both plakoglobin and E-cadherin were increased by AZA, and AZA enhanced their localization to sites of intercellular contact. Forced expression of plakoglobin and E-cadherin abrogated BeWo cell migration, indicative that repression of these genes was required for BeWo cell migration. Small interfering RNA-mediated depletion of the individual DNA methyltransferase (DNMT) molecules did not affect plakoglobin and E-cadherin promoter activity or BeWo cell migration. However, increases in plakoglobin and E-cadherin promoter activity and inhibition of BeWo cell migration was achieved with small interfering RNA-mediated depletion of both DNMT-3a and DNMT-3b. Epigenetic regulation of plakoglobin and E-cadherin is therefore pivotal for appropriate trophoblastic invasion in vitro.

Inhibition of GLI1 gene activation by Patched1.

Patched1 (PTCH1) is a human tumour suppressor that acts as an HH (Hedgehog) receptor protein and is important for embryonic patterning. PTCH1 mediates its effects through SMO (Smoothened) and represses the expression of HH target genes such as the transcription factor GLI1 (glioma 1) as well as PTCH1. Up-regulation of these genes has been observed in several cancer forms, including basal cell carcinoma, digestive track tumours and small cell lung cancer. The fact that PTCH1 down-regulates its own expression via 'negative feedback' is an important feature in HH signalling, as it keeps the balance between HH and PTCH1 activities that are essential for normal development. In the present study, we provide evidence that a novel mechanism allowing PTCH1 to maintain this balance may also exist. We show that gene activation by GLI1, the transcriptional effector of the pathway, can be down-regulated by PTCH1 without involvement of the canonical cascade of HH signalling events. Specifically, the SMO antagonist cyclopamine has no appreciable effects in blocking this PTCH1-mediated inhibition. Moreover, the negative GLI1 regulator SUFU (Suppressor of Fused) was also found to be dispensable. Additionally, deletion mapping of PTCH1 has revealed that the domains encompassed by amino acids 180-786 and 1058-1210 are of highest significance in inhibiting GLI1 gene activation. This contrasts with the importance of the PTCH1 C-terminal domain for HH signalling.

A novel first exon of the Patched1 gene is upregulated by Hedgehog signaling resulting in a protein with pathway inhibitory functions.

Patched homolog 1 (PTCH1) is a key component of the Hedgehog (HH) signaling pathway with three alternative first exons, but only exon 1B transcription depending on HH activation. Here, we show that in both human and mouse a novel PTCH1 first exon (1C) is expressed. Exon 1C transcription is upregulated by HH signaling, but the resulting PTCH1-1C protein has a lower capacity for pathway inhibition than PTCH1-1B.

Distinct roles of PTCH2 splice variants in Hedgehog signalling.

The human PTCH2 gene is highly similar to PTCH1, a tumour suppressor gene frequently mutated in basal cell carcinoma and several other tumour types. PTCH1 is a transmembrane protein believed to inhibit another transmembrane protein SMO (Smoothened), which mediates HH (Hedgehog) signalling. In this study, we analysed the biological properties of several PTCH2 splice variants. An mRNA form that lacked the last exon was abundantly expressed in all tissues examined, in contrast with the one that included it. Moreover, a transcript lacking exon 9, which is a part of a conserved sterol-sensing domain, was identified in intestine, prostate and cerebellum. In ovary, spleen, testis, cerebellum and skin, an mRNA lacking both exons 9 and 10 could also be observed. The different PTCH2 isoforms localized in the cytoplasm were capable of internalizing the N-terminal fragment of Sonic HH (Shh-N). Additionally, the PTCH2 gene was found to be a target of HH signalling. PTCH2 promoter regulation assays demonstrated that only one of the PTCH2 variants could inhibit the activity of SHH-N, whereas none was capable of inhibiting the activated form of SMO (SMO-M2) and this contrasts with PTCH1. Despite the fact that the PTCH2 isoforms lacked the ability to inhibit SMO-M2 activity, all PTCH2 variants as well as PTCH1, on co-transfection with Smo, were able to change Smo localization from being largely dispersed in the cytoplasm to the juxtanuclear region. Furthermore, the PTCH2 isoforms and PTCH1 co-localized in doubly transfected cells and an interaction between them was confirmed using immunoprecipitation assays. Using Ptch1-/- mouse cells, it was shown that the PTCH2 variants and PTCH1 differentially act to reconstitute not only the SHH but also the Desert HH-dependent transcriptional response. We conclude that in spite of their structural similarities, the PTCH2 isoforms have distinct functional properties when compared with PTCH1.

Alternative first exons of PTCH1 are differentially regulated in vivo and may confer different functions to the PTCH1 protein.

The PTCH1 gene is a human tumour suppressor gene frequently mutated in basal cell carcinoma (BCC) and several other tumour types. It encodes a receptor for soluble factors of the hedgehog family. Binding of hedgehog to the receptor relieves its inhibitory action on the transmembrane co-receptor Smoh. In this study we describe alternative first exons of the PTCH1 tumour suppressor gene and show that they are differentially regulated in normal tissues, exon 1B being expressed at very low levels and the major mRNA species containing exon 1 or 1A. Exon 1B transcripts were found to be specifically upregulated in nodular BCCs. The different PTCH1 transcripts all encode proteins that interact with Smoh in doubly transfected cells. Furthermore, functional assays demonstrated that whereas all PTCH1 isoforms can inhibit the activity of SHH, only the PTCH1B isoform is capable of fully inhibiting Smoh activity. The results indicate that in tumour cells the PTCH1B promoter is specifically activated and importantly, that the N-terminal part of PTCH1 including exon 1B is required for full inhibition of Smoh signaling but not for physical interaction with Smoh.