Association between the number of symptomatic mpox cases and the detection of mpox virus DNA in wastewater in Switzerland: an observational surveillance study.

AIM OF THE STUDY: The COVID-19 pandemic has drawn attention to the benefit of wastewater-based epidemiology, particularly when case numbers are underreported. Underreporting may be an issue with mpox, where biological reasons and stigma may prevent patients from getting tested. Therefore, we aimed to assess the validity of wastewater surveillance for monitoring mpox virus DNA in wastewater of a Central European city and its association with official case numbers. METHODS: Wastewater samples were collected between 1 July and 28 August 2022 in the catchment area of Basel, Switzerland, and the number of mpox virus genome copies they contained was determined by real-time quantitative PCR. Logistic regression analyses were used to determine the odds of detectability of mpox virus DNA in wastewater, categorised as detectable or undetectable. Mann-Whitney U tests were used to determine associations between samples that tested positive for the mpox virus and officially reported cases and patients' recorded symptomatic phases. RESULTS: Mpox virus DNA was detected in 15 of 39 wastewater samples. The number of positive wastewater samples was associated with the number of symptomatic cases (odds ratio [OR] = 2.18, 95% confidence interval (CI) = 1.38-3.43, p = 0.001). The number of symptomatic cases differed significantly between days with positive versus negative wastewater results (median = 11 and 8, respectively, p = 0.0024). CONCLUSION: Mpox virus DNA was detectable in wastewater, even when officially reported case numbers were low (0-3 newly reported mpox cases corresponding to 6-12 symptomatic patients). Detectability in wastewater was significantly associated with the number of symptomatic patients within the catchment area. These findings illustrate the value of wastewater-based surveillance systems when assessing the prevalence of emerging and circulating infectious diseases.

Influenza transmission dynamics quantified from RNA in wastewater in Switzerland.

INTRODUCTION: Influenza infections are challenging to monitor at the population level due to many mild and asymptomatic cases and similar symptoms to other common circulating respiratory diseases, including COVID-19. Methods for tracking cases outside of typical reporting infrastructure could improve monitoring of influenza transmission dynamics. Influenza shedding into wastewater represents a promising source of information where quantification is unbiased by testing or treatment-seeking behaviours. METHODS: We quantified influenza A and B virus loads from influent at Switzerland's three largest wastewater treatment plants, serving about 14% of the Swiss population (1.2 million individuals). We estimated trends in infection incidence and the effective reproductive number (Re) in these catchments during a 2021/22 epidemic and compared our estimates to typical influenza surveillance data. RESULTS: Wastewater data captured the same overall trends in infection incidence as laboratory-confirmed case data at the catchment level. However, the wastewater data were more sensitive in capturing a transient peak in incidence in December 2021 than the case data. The Re estimated from the wastewater data was roughly at or below the epidemic threshold of 1 during work-from-home measures in December 2021 but increased to at or above the epidemic threshold in two of the three catchments after the relaxation of these measures. The third catchment yielded qualitatively the same results but with wider confidence intervals. The confirmed case data at the catchment level yielded comparatively less precise R_e estimates before and during the work-from-home period, with confidence intervals that included one before and during the work-from-home period. DISCUSSION: Overall, we show that influenza RNA in wastewater can help monitor nationwide influenza transmission dynamics. Based on this research, we developed an online dashboard for ongoing wastewater-based influenza surveillance in Switzerland.

Extended-spectrum ß-lactamase-producing Enterobacterales in diverse foodstuffs: a prospective, longitudinal study in the city of Basel, Switzerland.

Background: The involvement of non-human-to-human transmission of extended-spectrum ß-lactamase-producing Enterobacterales (ESBL-PE) remains elusive. Foodstuffs may serve as reservoirs for ESBL-PE and contribute to their spread. Aim: We aimed to systematically investigate the presence and spatiotemporal distribution of ESBL-PE in diverse unprocessed foodstuffs of different origin purchased in a central European city. Methods: Chicken and green (herbs, salad, sprouts, vegetables) samples were collected monthly for two consecutive years, from June 2017 to June 2019, from large supermarket chains and small local food retailers, representing all ten postcode areas of the City of Basel (Switzerland), and the kitchen of the University Hospital Basel (Basel, Switzerland). After enrichment, presumptive ESBL-PE were isolated by selective culture methods and identified by Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. ESBL production was confirmed by phenotypic testing. Results: Among 947 food samples, 14.8% were positive for ESBL-PE isolate/s belonging to eight different ESBL-producing bacterial species. Escherichia coli and Serratia fonticola were predominant across samples (9 and 2%, respectively). Higher ESBL-PE prevalence was observed in chicken (25.9%) than in green (3.8%) samples (p < 0.001). Among greens, ESBL-PE were most frequently isolated from sprouts (15.2%). High ESBL-PE species diversity was observed among chicken samples, with E. coli as predominant (17.6%). ESBL-producing Enterobacter cloacae was detected among different greens. Yet, ESBL-producing Klebsiella pneumoniae was predominant in sprouts (12.1%). In total, 20.5% of samples from organic farming and 14.2% of samples from conventionally raised animals harbored an ESBL-producing isolate. Detection of ESBL-PE across samples differed between organic and non-organic when stratified by food source (p < 0.001), particularly among greens (12.5% organic, 2.4% conventional). High proportion of organic chicken samples was positive for ESBL-E. coli (33.3%), while the detection of several species characterized the conventional chicken samples. No significant differences in ESBL-PE frequences were detected between national (13.4%) and international samples (8.0%) (p = 0.122). Instead, differences were observed between regions of food production and countries (p < 0.001). No significant differences were found when comparing the proportion of ESBL-PE positive samples across districts, shop sizes and the hospital kitchen. The percentage of ESBL-PE positive samples did not differ monthly across the two-year sampling period (p = 0.107). Conclusion: Our findings indicate moderate dissemination of ESBL-PE in foodstuffs, especially between chicken products and sprouts. Chicken meat represents a source for several ESBL-producing Enterobacterales, especially E. coli, while greens are more prone to carry ESBL-K. pneumoniae and E. cloacae. We disclose the importance of food type, food production system and production origin when assessing the risk of contamination with different ESBL-PE species.

Spatiotemporal dissemination of ESBL-producing Enterobacterales in municipal sewer systems: a prospective, longitudinal study in the city of Basel, Switzerland.

Background: The contribution of community and hospital sources to the transmission of extended-spectrum ß-lactamase producing Enterobacterales (ESBL-PE) remains elusive. Aim: To investigate the extent of community dissemination and the contribution of hospitals to the spread of ESBL-PE by exploring their spatiotemporal distribution in municipal wastewater of the central European city of Basel. Methods: Wastewater samples were collected monthly for two consecutive years throughout Basel, Switzerland, including 21 sites across 10 postcode areas of the city collecting either community wastewater (urban sites, n = 17) or community and hospital wastewater (mixed sites, n = 4). Presumptive ESBL-PE were recovered by selective culture methods. Standard methodologies were applied for species identification, ESBL-confirmation, and quantification. Results: Ninety-five percent (477/504) of samples were positive for ESBL-PE. Among these isolates, Escherichia coli (85%, 1,140/1,334) and Klebsiella pneumoniae (11%, 153/1,334) were most common. They were recovered throughout the sampling period from all postcodes, with E. coli consistently predominating. The proportion of K. pneumoniae isolates was higher in wastewater samples from mixed sites as compared to samples from urban sites, while the proportion of E. coli was higher in samples from urban sites (p = 0.003). Higher numbers of colony forming units (CFUs) were recovered from mixed as compared to urban sites (median 3.2 × 102 vs. 1.6 × 102 CFU/mL). E. coli-counts showed moderate correlation with population size (rho = 0.44), while this correlation was weak for other ESBL-PE (rho = 0.21). Conclusion: ESBL-PE are widely spread in municipal wastewater supporting that community sources are important reservoirs entertaining the spread of ESBL-PE. Hospital-influenced abundance of ESBL-PE appears to be species dependent.

Quality of MALDI-TOF mass spectra in routine diagnostics: results from an international external quality assessment including 36 laboratories from 12 countries using 47 challenging bacterial strains.

OBJECTIVES: Matrix assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry (MS) is a widely used method for bacterial species identification. Incomplete databases and mass spectral quality (MSQ) still represent major challenges. Important proxies for MSQ are the number of detected marker masses, reproducibility, and measurement precision. We aimed to assess MSQs across diagnostic laboratories and the potential of simple workflow adaptations to improve it. METHODS: For baseline MSQ assessment, 47 diverse bacterial strains, which are challenging to identify by MALDI-TOF MS, were routinely measured in 36 laboratories from 12 countries, and well-defined MSQ features were used. After an intervention consisting of detailed reported feedback and instructions on how to acquire MALDI-TOF mass spectra, measurements were repeated and MSQs were compared. RESULTS: At baseline, we observed heterogeneous MSQ between the devices, considering the median number of marker masses detected (range = [2-25]), reproducibility between technical replicates (range = [55%-86%]), and measurement error (range = [147 parts per million (ppm)-588 ppm]). As a general trend, the spectral quality was improved after the intervention for devices, which yielded low MSQs in the baseline assessment as follows: for four out of five devices with a high measurement error, the measurement precision was improved (p-values <0.001, paired Wilcoxon test); for six out of ten devices, which detected a low number of marker masses, the number of detected marker masses increased (p-values <0.001, paired Wilcoxon test). DISCUSSION: We have identified simple workflow adaptations, which, to some extent, improve MSQ of poorly performing devices and should be considered by laboratories yielding a low MSQ. Improving MALDI-TOF MSQ in routine diagnostics is essential for increasing the resolution of bacterial identification by MALDI-TOF MS, which is dependent on the reproducible detection of marker masses. The heterogeneity identified in this external quality assessment (EQA) requires further study.

Wastewater monitoring of SARS-CoV-2 shows high correlation with COVID-19 case numbers and allowed early detection of the first confirmed B.1.1.529 infection in Switzerland: results of an observational surveillance study.

AIMS OF THE STUDY: Wastewater-based epidemiology has contributed significantly to the comprehension of the dynamics of the current COVID-19 pandemic. Its additional value in monitoring SARS-CoV-2 circulation in the population and identifying newly arising variants independently of diagnostic testing is now undisputed. As a proof of concept, we report here correlations between SARS-CoV-2 detection in wastewater and the officially recorded COVID-19 case numbers, as well as the validity of such surveillance to detect emerging variants, exemplified by the detection of the B.1.1.529 variant Omicron in Basel, Switzerland. METHODS: From July 1 to December 31, 2021, wastewater samples were collected six times a week from the inflow of the local wastewater treatment plant that receives wastewater from the catchment area of the city of Basel, Switzerland, comprising 273,075 inhabitants. The number of SARS-CoV-2 RNA copies was determined by reverse transcriptase-quantitative PCR. Spearman's rank correlation coefficients were calculated to determine correlations with the median seven-day incidence of genome copies per litre of wastewater and official case data. To explore delayed correlation effects between the seven-day median number of genome copies/litre wastewater and the median seven-day incidence of SARS-CoV-2 cases, time-lagged Spearman's rank correlation coefficients were calculated for up to 14 days. RNA extracts from daily wastewater samples were used to genotype circulating SARS-CoV-2 variants by next-generation sequencing. RESULTS: The number of daily cases and the median seven-day incidence of SARS-CoV-2 infections in the catchment area showed a high correlation with SARS-CoV-2 measurements in wastewater samples. All correlations between the seven-day median number of genome copies/litre wastewater and the time-lagged median seven-day incidence of SARS-CoV-2 cases were significant (p<0.001) for the investigated lag of up to 14 days. Correlation coefficients declined constantly from the maximum of 0.9395 on day 1 to the minimum of 0.8016 on day 14. The B.1.1.529 variant Omicron was detected in wastewater samples collected on November 21, 2021, before its official acknowledgement in a clinical sample by health authorities. CONCLUSIONS: In this proof-of-concept study, wastewater-based epidemiology proved a reliable and sensitive surveillance approach, complementing routine clinical testing for mapping COVID-19 pandemic dynamics and observing newly circulating SARS-CoV-2 variants.

Identification of a Cluster of Extended-spectrum Beta-Lactamase-Producing Klebsiella pneumoniae Sequence Type 101 Isolated From Food and Humans.

We report a cluster of extended-spectrum beta-lactamase (ESBL)-producing Klebsiella pneumoniae sequence type 101, derived from 1 poultry and 2 clinical samples collected within the setting of a prospective study designed to determine the diversity and migration of ESBL-producing Enterobacterales between humans, foodstuffs, and wastewater.

Transmission of ESBL-producing Enterobacteriaceae and their mobile genetic elements-identification of sources by whole genome sequencing: study protocol for an observational study in Switzerland.

INTRODUCTION: Extended-spectrum beta-lactamases (ESBL)-producing Enterobacteriaceae were first described in relation with hospital-acquired infections. In the 2000s, the epidemiology of ESBL-producing organisms changed as especially ESBL-producing Escherichia coli was increasingly described as an important cause of community-acquired infections, supporting the hypothesis that in more recent years ESBL-producing Enterobacteriaceae have probably been imported into hospitals rather than vice versa. Transmission of ESBL-producing Enterobacteriaceae is complicated by ESBL genes being encoded on self-transmissible plasmids, which can be exchanged among the same and different bacterial species. The aim of this research project is to quantify hospital-wide transmission of ESBL-producing Enterobacteriaceae on both the level of bacterial species and the mobile genetic elements and to determine if hospital-acquired infections caused by ESBL producers are related to strains and mobile genetic elements predominantly circulating in the community or in the healthcare setting. This distinction is critical in prevention since the former emphasises the urgent need to establish or reinforce antibiotic stewardship programmes, and the latter would call for more rigorous infection control. METHODS AND ANALYSIS: This protocol presents an observational study that will be performed at the University Hospital Basel and in the city of Basel, Switzerland. ESBL-producing Enterobacteriaceae will be collected from any specimens obtained by routine clinical practice or by active screening in both inpatient and outpatient settings, as well as from wastewater samples and foodstuffs, both collected monthly over a 12-month period for analyses by whole genome sequencing. Bacterial chromosomal, plasmid and ESBL-gene sequences will be compared within the cohort to determine genetic relatedness and migration between humans and their environment. ETHICS AND DISSEMINATION: This study has been approved by the local ethics committee (Ethikkommission Nordwest-und Zentralschweiz) as a quality control project (Project-ID 2017-00100). The results of this study will be published in peer-reviewed medical journals, communicated to participants, the general public and all relevant stakeholders.

Monitoring of genetically modified Escherichia coli in laboratory wastewater.

Containment of genetically modified (GM) microorganisms such as Escherichia coli is a legal requirement to protect the environment from an unintended release and to avoid horizontal gene transfer (HGT) of recombinant DNA to native bacteria. In this study, we sampled the laboratory wastewater (LWW) at a large Swiss university from three sources over 2 years and cultured ampicillin-resistant, presumptive GM E. coli. From a total of 285 samples, 127 contained presumptive GM E. coli (45%) at a mean concentration of 2.8 × 102 CFU/ml. Plasmid DNA of 11 unique clones was partially or entirely sequenced. All consisted of cloning vectors harboring research-specific inserts. To estimate the chance of HGT between GM E. coli and native bacteria in LWW, we identified taxa representative for the bacterial community in LWW using 16S rRNA amplicon sequencing and measured conjugation frequencies of E. coli with five LWW isolates. At optimal conjugation conditions, frequencies were between 3.4 × 10-3 and 2.4 × 10-5. Given the absence of transferable broad-host range plasmids and suboptimal conjugation conditions in the LWW system, we conclude that the chance of HGT is relatively low. Still, this study shows that the implementation of robust containment measures is key to avoid the escape of GM microorganisms.

Draft Genome Sequence of Klebsiella pneumoniae 704SK6, an OXA-48- and CTX-M-15-Encoding Wastewater Isolate.

The Swiss wastewater isolate Klebsiella pneumoniae 704SK6, encoding OXA-48 and CTX-M-15 ß-lactamases, was fully sequenced. The assembly resulted in an open chromosome of 5,208,104 bp in size (G+C content, 57.6%) and four closed plasmid sequences of 209,651, 197,670, 65,998, and 63,605 bp in size.

Complete Genome Sequence of Enterobacter cloacae 704SK10, an OXA-48-Encoding Wastewater Isolate.

Here we present the complete genome sequence of Enterobacter cloacae 704SK10, a Swiss wastewater isolate encoding an OXA-48 carbapenemase. Assembly resulted in closed sequences of the 4,876,946-bp chromosome, a 111,184-bp IncF plasmid, and an OXA-48-encoding IncL plasmid (63,458 bp) nearly identical to the previously described plasmid pOXA-48.

Complete Genome Sequence of Citrobacter freundii 705SK3, an OXA-48-Encoding Wastewater Isolate.

We present the genome sequence of Citrobacter freundii 705SK3, a wastewater isolate harboring an IncL OXA-48-encoding plasmid. Assembly of the genome resulted in a 5,242,839-bp circular chromosome (GC content, 52%) and two closed plasmids of 296,175 bp and 63, 458 bp in size.

Complete Genome Sequence of Escherichia coli ABWA45, an rmtB-Encoding Wastewater Isolate.

We present the complete genome sequence of Escherichia coli ABWA45, a 16S rRNA methyltransferase-producing wastewater isolate. Assembly and annotation resulted in a 5,094,639-bp circular chromosome and four closed plasmids of 145,220 bp, 113,793 bp, 57,232 bp, and 47,900 bp in size. Furthermore, a small open plasmid (7,537 bp in size) was assembled.

Wastewater is a reservoir for clinically relevant carbapenemase- and 16s rRNA methylase-producing Enterobacteriaceae.

The aim of this study was to evaluate wastewater for carbapenemase-producing Enterobacteriaceae (CPE) and 16S rRNA methylase-producing Gram-negative bacteria (MPB) and to assess their occurrence following wastewater treatment. Wastewater samples were collected between June 2015 and March 2016 in the sewage network of the city of Basel (Switzerland) from sites located before and after influx of wastewater from the hospital into the sewage network. Samples were also obtained from the influent and effluent of the receiving wastewater treatment plant. Samples were screened for CPE and MPB using selective media. Escherichia coli and Klebsiella pneumoniae were typed by multilocus sequence typing (MLST). Carbapenemase and 16S rRNA methylase genes were identified by PCR and sequencing. Resistance profiles were determined by the disk diffusion test and Etest. The occurrence of CPE and MPB was increased downstream of hospital wastewater influx. Of 49 CPE isolates, 9 belonged to OXA-48-producing E. coli clone D:ST38, 7 were OXA-48-producing Citrobacter freundii, and 6 were KPC-2- or OXA-48-producing K. pneumoniae belonging to clonal complex 258. NDM (NDM-1, -5 and -9) and VIM (VIM-1) producers were detected sporadically. MPB included ArmA- and RmtB-producing E. coli and Citrobacter spp. Isolates corresponding to strains from wastewater were detected in the effluent of the treatment plant. Conclusively, CPE and MPB, predominantly OXA-48-producing Enterobacteriaceae, are readily detected in wastewater, survive wastewater treatment and are released into the aquatic environment. OXA-48-producers may represent an emerging threat to public health and environmental integrity.

Low level impurities in imported wheat are a likely source of feral transgenic oilseed rape (Brassica napus L.) in Switzerland.

In Switzerland, the cultivation of genetically modified (GM) oilseed rape (Brassica napus L.) and the use of its seeds for food and feed are not permitted. Nevertheless, the GM oilseed rape events GT73, MS8×RF3, MS8 and RF3 have recently been found in the Rhine port of Basel, Switzerland. The sources of GM oilseed rape seeds have been unknown. The main agricultural good being imported at the Rhine port of Basel is wheat and from 2010 to 2013, 19% of all Swiss wheat imports originated from Canada. As over 90% of all oilseed rape grown in Canada is GM, we hypothesised that imports of Canadian wheat may contain low level impurities of GM oilseed rape. Therefore, waste fraction samples gathered during the mechanical cleaning of Canadian wheat from two Swiss grain mills were analysed by separating oilseed rape seeds from waste fraction samples and testing DNA of pooled seeds for the presence of transgenes by real-time PCR. Furthermore, oilseed rape seeds from each grain mill were sown in a germination experiment, and seedling DNA was tested for the presence of transgenes by real-time PCR. GT73, MS8×RF3, MS8 and RF3 oilseed rape was detected among seed samples and seedlings of both grain mills. Based on this data, we projected a mean proportion of 0.005% of oilseed rape in wheat imported from Canada. Besides Canadian wheat, the Rhine port of Basel does not import any other significant amounts of agricultural products from GM oilseed rape producing countries. We therefore conclude that Canadian wheat is the major source of unintended introduction of GM oilseed rape seeds into Switzerland.

Unexpected diversity of feral genetically modified oilseed rape (Brassica napus L.) despite a cultivation and import ban in Switzerland.

Despite cultivation and seed import bans of genetically modified (GM) oilseed rape (Brassica napus L.), feral GM plants were found growing along railway lines and in port areas at four sites in Switzerland in 2011 and 2012. All GM plants were identified as glyphosate-resistant GM event GT73 (Roundup Ready, Monsanto). The most affected sites were the Rhine port of Basel and the St. Johann freight railway station in Basel. To assess the distribution and intra- and interspecific outcrossing of GM oilseed rape in more detail, we monitored these two sites in 2013. Leaves and seed pods of feral oilseed rape plants, their possible hybridization partners and putative hybrid plants were sampled in monthly intervals and analysed for the presence of transgenes by real-time PCR. Using flow cytometry, we measured DNA contents of cell nuclei to confirm putative hybrids. In total, 2787 plants were sampled. The presence of GT73 oilseed rape could be confirmed at all previously documented sampling locations and was additionally detected at one new sampling location within the Rhine port. Furthermore, we found the glufosinate-resistant GM events MS8xRF3, MS8 and RF3 (all traded as InVigor, Bayer) at five sampling locations in the Rhine port. To our knowledge, this is the first time that feral MS8xRF3, MS8 or RF3 plants were detected in Europe. Real-time PCR analyses of seeds showed outcrossing of GT73 into two non-GM oilseed rape plants, but no outcrossing of transgenes into related wild species was observed. We found no hybrids between oilseed rape and related species. GM plants most frequently occurred at unloading sites for ships, indicating that ship cargo traffic is the main entry pathway for GM oilseed rape. In the future, it will be of major interest to determine the source of GM oilseed rape seeds.

Detection of feral GT73 transgenic oilseed rape (Brassica napus) along railway lines on entry routes to oilseed factories in Switzerland.

To obtain a reference status prior to cultivation of genetically modified oilseed rape (OSR, Brassica napus L.) in Switzerland, the occurrence of feral OSR was monitored along transportation routes and at processing sites. The focus was set on the detection of (transgenic) OSR along railway lines from the Swiss borders with Italy and France to the respective oilseed processing factories in Southern and Northern Switzerland (Ticino and region of Basel). A monitoring concept was developed to identify sites of largest risk of escape of genetically modified plants into the environment in Switzerland. Transport spillage of OSR seeds from railway goods cars particularly at risk hot spots such as switch yards and (un)loading points but also incidental and continuous spillage were considered. All OSR plants, including their hybridization partners which were collected at the respective monitoring sites were analyzed for the presence of transgenes by real-time PCR. On sampling lengths each of 4.2 and 5.7 km, respectively, 461 and 1,574 plants were sampled in Ticino and the region of Basel. OSR plants were found most frequently along the routes to the oilseed facilities, and in larger amounts on risk hot spots compared to sites of random sampling. At three locations in both monitored regions, transgenic B. napus line GT73 carrying the glyphosate resistance transgenes gox and CP4 epsps were detected (Ticino, 22 plants; in the region of Basel, 159).

The intracellular domain of teneurin-2 has a nuclear function and represses zic-1-mediated transcription.

Teneurin-2, a vertebrate homologue of the Drosophila pair-rule gene ten-m/odz, is revealed to be a membrane-bound transcription regulator. In the nucleus, the intracellular domain of teneurin-2 colocalizes with promyelocytic leukemia (PML) protein in nuclear bodies implicated in transcription control. Since Drosophila ten-m acts epistatically to another pair-rule gene opa, we investigated whether gene regulation by the mammalian opa homologue zic-1 was influenced by the intracellular domain of teneurin-2. We found that zic-mediated transcription from the apolipoprotein E promoter was inhibited. Release of the intracellular domain of teneurin-2 could be stimulated by homophilic interaction of the extracellular domain, and the intracellular domain was stabilized by proteasome inhibitors. We have previously shown that teneurin-2 is expressed by neurons belonging to the same functional circuit. Therefore, we hypothesize that homophilic interaction enables neurons to identify their targets and that the release of the intracellular domain of teneurin-2 provides them with a signal to switch their gene expression program from growth towards differentiation once the proper contact has been made.