Cost-effectiveness of anti-SARS-CoV-2 antibody diagnostic tests in Brazil.

BACKGROUND: Although serologic tests for COVID-19 diagnosis are rarely indicated nowadays, they remain commercially available and widely used in Brazil. The objective of this study was to evaluate the cost-effectiveness of anti-SARS-CoV-2antibody diagnostic tests for COVID-19 in Brazil. METHODS: Eleven commercially available diagnostic tests, comprising five lateral-flow immunochromatographic assays (LFAs) and six immunoenzymatic assays (ELISA) were analyzed from the perspective of the Brazilian Unified Health System. RESULTS: The direct costs of LFAs ranged from US$ 11.42 to US$ 17.41and of ELISAs, from US$ 6.59 to US$ 10.31. Considering an estimated disease prevalence between 5% and 10%, the anti-SARS-CoV-2 ELISA (IgG) was the most cost-effective test, followed by the rapid One Step COVID-19 Test, at an incremental cost-effectiveness ratio of US$ 2.52 and US$ 1.26 per properly diagnosed case, respectively. Considering only the LFAs, at the same prevalence estimates, two tests, the COVID-19 IgG/IgM and the One Step COVID-19 Test, showed high effectiveness at similar costs. For situations where the estimated probability of disease is 50%, the LFAs are more costly and less effective alternatives. CONCLUSIONS: Nowadays there are few indications for the use of serologic tests in the diagnosis of COVID-19 and numerous commercially available tests, with marked differences are observed among them. In general, LFA tests are more cost-effective for estimated low-COVID-19-prevalences, while ELISAs are more cost-effective for high-pretest-probability scenarios.

A cell-free high throughput assay for assessment of SARS-CoV-2 neutralizing antibodies.

Highly accurate serological tests are key to assessing the prevalence of SARS-CoV-2 antibodies and the level of immunity in the population. This is important to predict the current and future status of the pandemic. With the recent emergence of new and more infectious SARS-CoV-2 variants, assays allowing for high throughput analysis of antibodies able to neutralize SARS-CoV-2 become even more important. Here, we report the development and validation of a robust, high throughput method, which enables the assessment of antibodies inhibiting the binding between the SARS-CoV-2 spike protein and angiotensin converting enzyme 2 (ACE2). The assay uses recombinantly produced spike-f and ACE2 and is performed in a bead array format, which allows analysis of up to 384 samples in parallel per instrument over seven hours, demanding only one hour of manual handling. The method is compared to a microneutralization assay utilising live SARS-CoV-2 and is shown to deliver highly correlating data. Further, a comparison with a serological method that measures all antibodies recognizing the spike protein shows that this type of assessment provides important insights into the neutralizing efficiency of the antibodies, especially for individuals with low antibody levels. This method can be an important and valuable tool for large-scale assessment of antibody-based neutralization, including neutralization of new spike variants that might emerge.

Simple workflow to repurpose SARS-CoV-2 swab/serum samples for the isolation of cost-effective antibody/antigens for proteotyping applications and diagnosis.

Supply shortage for the development and production of preventive, therapeutic, and diagnosis tools during the COVID-19 pandemic is an important issue affecting the wealthy and poor nations alike. Antibodies and antigens are especially needed for the production of immunological-based testing tools such as point-of-care tests. Here, we propose a simple and quick magnetic nanoparticle (MNP)-based separation/isolation approach for the repurposing of infected human samples to produce specific antibodies and antigen cocktails. Initially, an antibody cocktail was purified from serums via precipitation and immunoaffinity chromatography. Purified antibodies were conjugated onto MNPs and used as an affinity matrix to separate antigens. The characterization process was performed by ELISA, SDS-PAGE, electrochemistry, isothermal titration calorimetry, and LC-Q-TOF-MS/MS analyses. The MNP-separated peptides can be used for mass spectrometry-based as well as paper-based lateral flow assay diagnostic. The exploitation of the current workflow for the development of efficient diagnostic tools, specific treatments, and fundamental research can significantly impact the present or eventual pandemic. This workflow can be considered as a two birds, one stone-like strategy.

A study of rural chicken farmers, diseases and remedies in the Eastern Cape province of South Africa.

The source of emerging diseases and antimicrobial resistance is of increasing interest to epidemiologists. This paper looks at village chickens as such a source. In addition, infectious diseases constitute a major challenge to the growth and profitability of the rural poultry sector in Sub-Saharan Africa. A serological survey was conducted to estimate the apparent seroprevalence of selected chicken diseases in the Eastern Cape Province of South Africa alongside a sociological survey of poultry farmers and the remedies most commonly used to prevent diseases in their flocks. Sera collected from village chickens (n = 1007) in the province were screened for specific antibodies against Newcastle disease (ND), avian influenza (AI), avian infectious bronchitis (IB) and Mycoplasma gallisepticum (MG). The overall seroprevalence of ND, AI, IB and MG in the province was found to be 69.2 % (95 % CI 51.9-86.5%); 1.8 % (95 % CI 0.2-3.4%); 78.5 % (95 % CI 74.9-82%) and 55.8 % (95 % CI 41.3-70.3%) respectively with clustering found at the District level. Cross hemagglutination inhibition (HI) tests indicated that the chickens were exposed to the ND vaccine. AI ELISA-positive samples were tested using HIs against the H5, H6 and H7-subtypes, but only H6-specific antibodies were detected. Avian influenza strains shared the common ancestor responsible for the 2002 chicken outbreak in KwaZulu-Natal Province. The majority of chicken farmers were females and pensioners (69 % and 66.1 % respectively) and had a primary school education (47.1 %). Traditional remedies were commonly used by farmers (47.15 %) and among the remedies, Aloe plant (Aloe ferox Mill.) or ikhala (Xhosa) was the most commonly used product (28.23 %) for preventing and reducing mortalities among village chickens. The findings stress the importance of village chickens as a substitute for social welfare and highlight the exposure of village chickens to important chicken pathogens. The economic impact of these pathogens on the development of this sub-sector needs further investigation. Village chickens are a potential source of virulent Newcastle disease virus (NDV) because of the lack of vaccination and biosecurity. They may serve as amplification hosts which increases the probability that virulent NDV could spill over into commercial poultry flocks due to large amounts of circulating virus. The zoonotic threat of circulating H6N2 viruses raise concern due to their mutation and reassortment among chickens and a potential movement of infected birds within the province. Finally, the use of antibiotics by untrained chicken farmers constitute another major concern as it could serve as a source of antimicrobial resistance (AMR).

Genetic diversity, haplotype analysis, and risk factor assessment of hepatitis A virus isolates from the West Bank, Palestine during the period between 2014 and 2016.

BACKGROUND: Hepatitis A virus (HAV) infection is one of the major causes of acute viral hepatitis. HAV genotypes and its genetic diversity is rarely investigated in our region as well as worldwide. AIMS: The aims of the present study were to determine the HAV genotypes and its risk factors and to investigate the genetic diversity of the HAV isolates in the West Bank, Palestine. STUDY DESIGN: A cohort of 161 clinically and laboratory-confirmed HAV (IgM-positive) cases and 170 apparently healthy controls from all the districts of the West Bank, Palestine during the period of 2014 to 2016 were tested for HAV infection using IgM antibodies, RT-PCR and sequence analysis of the VP3/VP1 junction region of the HAV genome. Phylogenetic analysis, genetic diversity and haplotypes analysis were used to characterize the VP3/VP1 sequences. RESULTS: All the 34 sequences of the HAV were found to be of HAV-IB sub-genotype. The phylogenetic analysis showed four main clusters with cluster III exclusively consisting of 18 Palestinian isolates (18/23-78%), but with weak bootstrap values. A high haplotype diversity (Hd) and low nucleotide diversity (π) were observed. Cluster III showed high number of haplotypes (h = 8), but low haplotype (gene) diversity (Hd = 0.69). A total of 28 active haplotypes with some consisting of more than one sequence were observed using haplotype network analysis. The Palestinian haplotypes are characterized by closely related viral haplotypes with one SNV away from each other which ran parallel to cluster III in the phylogenetic tree. A smaller Palestinian haplotype (4 isolates) was three SNVs away from the major haplotype cluster (n = 10) and closer to others haplotypes from Iran, Spain, and South Africa. Young age, low level of parent's education, infrequent hand washing before meals, and drinking of un-treated water were considered the major HAV risk factors in the present study. CONCLUSION: Haplotype network analysis revealed haplotype variation among the HAV Palestinian sequences despite low genetic variation and nucleotide diversity. In addition, this study reconfirmed that age and parent's level of education as HAV risk factors, while hand washing and treating drinking water as protective factors.

Assessment of diagnostic and analytic performance of the SD Bioline Dengue Duo test for dengue virus (DENV) infections in an endemic area (Savannakhet province, Lao People's Democratic Republic).

BACKGROUND: Rapid tests detecting both dengue virus (DENV) NS1 antigen and anti-DENV IgM and IgG antibodies facilitate diagnosis of dengue fever (DF) in resource-poor settings. METHODOLOGY/PRINCIPAL FINDINGS: 92 acute phase serum samples from patients with a PCR-confirmed DENV infection collected in Lao People's Democratic Republic (Lao PDR) in 2013 and 2015 were analyzed with the SD Bioline Dengue Duo test. A subset of 74 samples was additionally tested with the Platelia NS1 antigen test, the Panbio DENV µ-capture ELISA and the Panbio DENV IgG ELISA. IgM seroconversion was assayed using follow-up samples of 35 patients collected in the convalescent phase. 57.6%, 22.8% and 44.6% of acute phase serum samples tested positive in the SD Bioline Dengue Duo NS1, IgM, and IgG test, respectively. Diagnostic sensitivity of the SD Bioline Dengue Duo NS1 test strongly correlated with viral load, decreased rapidly over the acute phase of the disease, and was significantly reduced in presence of high anti-DENV IgG antibody titers resulting from secondary DENV infection. While a good concordance (Cohen's kappa 0.78) was found between the SD Bioline Dengue Duo NS1 test and the Platelia NS1 antigen ELISA, both the SD Bioline Dengue Duo IgM and IgG test displayed a significantly lower sensitivity than the corresponding ELISA tests. CONCLUSIONS/SIGNIFICANCE: The SD Bioline Dengue Duo test is a valuable tool for diagnosis of DENV infections especially when analyzing early acute phase samples with high viral load. Nevertheless, in endemic areas, where secondary flavivirus infections are common, diagnostic sensitivity of the NS1 and IgM test components may be compromised.

Assessment on reticuloendotheliosis virus infection in specific-pathogen-free chickens based on detection of yolk antibody.

Reticuloendotheliosis virus (REV) is the most frequent exogenous virus that contaminates attenuated vaccines. Therefore, it is extremely important to select REV-free specific-pathogen-free (SPF) chicken embryos. Generally, REV infection is assessed by detecting REV antibodies in SPF chickens. This present study seeks to evaluate REV infection by replacing serum antibody detection with yolk antibody detection. A cohort of 40 nineteen-week-old SPF chickens were artificially inoculated with REV, with 32 SPF chickens raised in another isolation environment served as a blank control. Eggs and serum from 23-week-old chickens were sampled, and yolks were diluted separately to ratios of 1:150, 1:200, 1:300 and 1:400, which were detected together with serum. We found that the yolk antibody detection findings at a dilution of 1:300 had the highest coincidence rate compared with that based on serum antibody measurements. At a dilution ratio of 1:300 for yolk antibody, 72 chickens were continuously observed for 10 weeks from 25- to 34-weeks-old. Our findings were based on serum antibody or yolk antibody detection, and the evaluation results were completely consistent. Therefore, all serum antibody-positive chickens were yolk antibody-positive, and vice versa. Accordingly, vaccine producers can estimate REV cleanliness in a poultry farm by sampling yolk antibody titers.

A simple, inexpensive, robust and sensitive dot-blot assay for equal detection of the nonstructural-1 glycoprotein of all dengue virus serotypes.

BACKGROUND: Detection of dengue virus (DENV) soluble/excreted (s/e) form of the nonstructural-1 (NS1) glycoprotein in patient acute-phase sera is ideal for diagnosis. The commercially-available detection assays are, however, too expensive for routine use and have low specificity, particularly for the s/e NS1 glycoprotein of DENV-2 and DENV-4, which are important causes of lethal human disease worldwide. METHODS: Mouse monoclonal antibodies (MAbs) were generated and screened against s/e NS1 glycoprotein purified from each DENV serotype to obtain those that reacted equally with each serotype, but not with yellow fever virus (YFV) s/e NS1 glycoprotein or human serum proteins. One MAb, MAb 2C4.6, was further tested against these DENV glycoproteins in human sera using simple, peroxidase-labelled secondary antibody/substrate-developed dot-blot assays. RESULTS: Optimal quenching of endogenous human serum peroxidases was attained using 3% H(2)O(2) in H(2)0 for 5 min. MAb 2C4.6 showed an acceptable detection sensitivity of < 32 ng/ml for the s/e NS1 glycoprotein of each DENV serotype but did not cross-react with the YFV s/e NS1 glycoprotein or human serum proteins. By contrast, the LX1 epitope-specific MAb, 3D1.4, showed similar detection sensitivity against only the DENV-1 NS1 glycoprotein, consistent with results from commercial DENV s/e NS1 glycoprotein detection assays.DENV s/e NS1 glycoproteins were stable in human sera after drying on the nitrocellulose membranes and storage for one month at ambient temperature (28°C) before being processed. The total assay time was reduced to 3 h without any loss of detection sensitivity. This dot-blot format was ideal for the circulating immune complex disruption step, which is required for increased DENV s/e NS1 glycoprotein detection. CONCLUSIONS: This is the first study to determine the detection sensitivity of MAbs against known concentrations of s/e NS1 glycoprotein from each DENV serotype. The preparation of patient serum samples for dot-blot assays can be performed by staff with a basic level of training and storage at low temperatures (e.g., -80°C) is not necessary. These simple, inexpensive (US$ 0.05/sample), robust, sensitive and relatively rapid assays, using improved MAbs such as MAb 2C4.6, should be ideal for the diagnosis of all DENV serotypes in DENV endemic regions.

A dimerized single-chain variable fragment system for the assessment of neutralizing activity of phage display-selected antibody fragments specific for cytomegalovirus.

Cytomegalovirus (CMV) causes severe sequelae in congenitally infected newborns and may cause life-threatening disease in immuno-deficient patients. Recent findings demonstrate the possibility to alleviate the disease by infusing intravenous immunoglobulin G (IgG) preparations, indicating that antibodies are an effective therapeutic option. Modern molecular methodologies, like phage display, allow for the development of specific antibodies targeting virtually any antigen, including those of CMV. However, such methodologies do not in general result in products that by themselves mediate biological activity. To facilitate a semi-high-throughput approach for functional screening in future efforts to develop efficacious antibodies against CMV, we have integrated two different approaches to circumvent potential bottlenecks in such efforts. Firstly, we explored an approach that permits easy transfer of antibody fragment encoding genes from commonly used phage display vectors into vectors for the production of divalent immunoglobulins. Secondly, we demonstrate that such proteins can be applied in a novel reporter-based neutralization assay to establish a proof-of-concept workflow for the generation of neutralizing antibodies against CMV. We validated our approach by showing that divalent antibodies raised against the antigenic domain (AD)-2 region of gB effectively neutralized three different CMV strains (AD169, Towne and TB40/E), whereas two antibodies against the AD-1 region of gB displayed minor neutralizing capabilities. In conclusion, the methods investigated in this proof-of-concept study enables for a semi-high-throughput workflow in the screening and investigation of biological active antibodies.

Assessment of sensitivity and specificity of first, second, and third generation EIA for the detection of antibodies to HIV-1 in oral fluid.

The performances of three blood-based immunoassays test kits were compared with regard to their ability to detect HIV-1 antibody in oral fluid. It was found that these three kits differ in their ability to detect HIV-1 antibody. Notably, a third generation EIA which has been shown to possess superior sensitivity for antibody detection in plasma appears to possess no sensitivity advantage for detecting HIV-1 antibody in oral fluid.

Seroepidemiology of varicella and the reliability of a self-reported history of varicella infection in Singapore military recruits.

INTRODUCTION: Varicella is an acute disease with significant morbidity. However, there is little knowledge on the seroepidemiology of the disease in Singapore. The objective of this study was to assess the seroprevalence of varicella zoster virus (VZV) antibodies in military recruits in Singapore and to ascertain the predictive value of a self-reported history of varicella. The latter is a possible proxy for seroprevalence, and may be used to provide efficient identification of candidates for vaccination. MATERIALS AND METHODS: From September 2000 to October 2005, 2189 servicemen were selected during their pre-enlistment medical check-up. Blood samples were obtained to determine the varicella IgG levels via enzyme-linked immunosorbent assay (ELISA). Information about the participant's race, history of varicella and vaccination, and other clinical variables were obtained through a questionnaire. RESULTS: The overall prevalence of VZV seropositivity in military recruits was 76.0% (75.8% in the 16 years to 20 years age group). For the reported history, 73.7% of Chinese participants, 73.0% of Malays, and 63.6% of Indians reported having had varicella infection and/or vaccination. Overall, the sensitivity, specificity, positive and negative predictive values of a self-reported history of varicella for serologically confirmed immunity were 87.2%, 83.2%, 94.3% and 67.1% respectively. CONCLUSIONS: The prevalence of VZV antibodies in pre-enlistees to the Singapore Armed Forces (SAF) is high. Incidence of varicella in the SAF is on the wane, indicating an increase in herd immunity against VZV. A recalled history of varicella infection was also a good predictor of serological immunity and may be used for selection for vaccination.

Declining trend in the seroprevalence of infection with hepatitis A virus in Thailand.

Since the mid 1970s, infection with hepatitis A virus (HAV) in Thailand has shifted from hyper-endemic to mesoendemic. In 2004, to explore this trend in prevalence further, 3997 subjects from four geographically distinct provinces of Thailand were tested, in a commercial ELISA, for antibodies to HAV. The results indicate that the seroprevalence of HAV continues to fall, almost certainly because the profound socio-economic development that has occurred over the last few decades in Thailand has brought with it significant improvements in sanitation and personal hygiene. As exposure to HAV declines, however, the risks of symptomatic and potentially severe infection in adulthood (rather than asymptomatic infection during childhood) and of epidemics of such infection, which would lead to profound economic loss, increases. Improvements in hygiene and sanitation to reduce exposure to the virus and measures to reduce the incidence of symptomatic disease in those infected, such as vaccination (which may only be cost-effective when targeted at high-risk groups), need to be carefully considered.

Assessment of the use of gross lesions at post-mortem to detect outbreaks of classical swine fever.

The performance of pathological findings as a diagnostic tool for the detection of classical swine fever (CSF) outbreaks during the 1997/1998 CSF-epidemic in The Netherlands was evaluated by constructing and analysing receiver operating characteristic (ROC) curves. This was done at the individual pig level and at the submission level (a group of pigs from the same herd submitted together for post-mortem investigation). At post-mortem examination, the tonsils, spleen, ileo-caecal valve and renal pelvis were sampled, sent to the reference laboratory, and tested by means of a CSF-specific fluorescent antibody test in combination with a confirmatory test. This resulted in an infection status at the individual pig level. The infection status and pathological findings of 1072 individual pigs from a total of 230 infected herds were included in this analysis. We also included submissions of pigs from herds that were sent to post-mortem examination because of a clinically CSF-suspect situation but afterwards were concluded to be from non-infected herds. Infection status and pathological findings of 1224 individual pigs from a total of 241 non-infected herds were included in the analysis. Pneumonia, pleuritis, chronic bronchitis, pulmonary oedema, chronic gastric ulceration, dry faecal contents in the colon, conjunctivitis, haemorrhages in the renal pelvis, renal haemorrhages, splenic enlargement, haemorrhages in the urinary bladder, haemorrhagic and enlarged lymph nodes were the most frequently recorded pathological findings during a post-mortem examination of pigs submitted in a CSF-suspect clinical situation. However, some of these pathological findings (e.g. pneumonia, pleuritis) were almost evenly distributed in infected and in non-infected pigs, resulting in a high sensitivity combined with a low specificity. The area under the ROC curve of pathological findings at the individual pig level and at the submission level was 0.720 and 0.782, respectively, which was significantly (P<0.0001) larger than the area under the random ROC curve. It was concluded that, although gross pathology is a legitimate test, its quantitative contribution to the detection of CSF is limited.

Clinical manifestations and laboratory assessment in an enterovirus 71 outbreak in southern Taiwan.

An epidemic of enterovirus 71 (EV71) infection compatible with hand, foot and mouth disease and associated with high morbidity and mortality occurred in Taiwan in 1998. We recruited 90 patients (50 males, 40 females) with definite EV71 infections for clinical and laboratory analysis. The neurological signs and symptoms, all of which occurred during the febrile period, in patients with central nervous system (CNS) involvement (aseptic meningitis, encephalitis or myelitis) were myoclonic jerks (23/33), vomiting (10/33), ataxia (7/33), lethargy (6/33), seizure (4/33) and tremor (2/33). Patients with CNS involvement had longer durations of fever (4.6+/-0.2 vs. 3.1+/-0.3 d; p <0.01) and a higher white blood cell count (12,512+/-658 vs. 10,607+/-409 cells/mm3; p = 0.01) than patients without CNS involvement. The case fatality rate in patients with CNS involvement was 4/33 (12%), whereas no fatalities (0/57) occurred in patients without CNS involvement. Six of 11 patients subjected to MRI showed a high intensity T2-weighted signal in the brainstem. A nested fluorescent RT-PCR for detection of virus in throat and stool specimens showed higher sensitivity than viral culture. Viremia was detectable using RT-PCR in 20% of cases (3/15), whereas no virus was isolated from culture or detected by RT-PCR in cerebrospinal fluid.

A retrospective analysis of the infectious bovine rhinotracheitis (bovine herpes virus-1) surveillance program in Norway using Monte Carlo simulation models.

Serological surveillance for antibodies against bovine herpes virus type I (BHV-1) which causes infectious bovine rhinotracheitis and infectious pustular vulvovaginitis has been carried out since 1992 in Norway. Since 1993 (when a single infected herd was detected) all bulk-milk and pooled-serum samples have been negative for BHV-1 antibodies. This paper describes the use of Monte Carlo simulation models for the analysis and interpretation of the results of the surveillance and provides support for the contention that the Norwegian cattle population is not infected by BHV-1.

Use and cost of hospital and community service provision for children with HIV infection at an English HIV referral centre.

OBJECTIVE: To describe the use of hospital and community services for children infected with HIV and estimate the cost per patient-year by stage of HIV infection during the era of antiretroviral monotherapy. DESIGN: Data on the use of hospital services were collected from case notes; the use of statutory and nonstatutory community services was recorded through diaries and interviews. Total cost estimates were calculated from unit costs from relevant hospital departments and community organisations. SETTING: Children managed at St. Mary's Hospital (London, England) between 1 January 1986 and 31 December 1994, some of whom used statutory and nonstatutory community services in South East England between 1 November 1994 and 31 May 1996. PATIENTS AND PARTICIPANTS: 118 children with positive HIV antibody status. MAIN OUTCOME MEASURES AND RESULTS: Mean inpatient days, outpatient visits, tests and procedures performed, drugs prescribed, community services used, associated unit costs and average cost estimates per patient-year by stage of HIV infection (1995/1996 values), and lifetime costs. Service provision during the study period was predominantly hospital-based. The use of services increased for different stages of HIV infection and increased with increasing severity of HIV infection. A shift from an inpatient-based to an outpatient-based service was seen between the periods 1986 to 1991 and 1992 to 1994. As symptoms evolved, children used more hospital inpatient services, with an accompanying shift in the use of community services from general services, such as schooling, to increased use of nurses, social care and home help. The estimated total cost of hospital and community care was 18,600 Pounds per symptomatic non-AIDS patient per year and 46,600 Pounds per AIDS patient per year. Similar estimates for children with indeterminate HIV infection and asymptomatic infection amounted to 8300 Pounds and 4800 Pounds per patient-year, respectively. Nondiscounted lifetime costs for hospital care amounted to 152,400 Pounds (44,300 Pounds to 266,800 Pounds) compared with discounted lifetime costs of 122,700 Pounds (42,000 Pounds to 182,200 Pounds); nondiscounted lifetime costs for community care amounted to 24,300 Pounds (7900 Pounds to 41,600 Pounds) compared with discounted lifetime costs of 21,000 Pounds (6800 Pounds to 32,000 Pounds). CONCLUSIONS: The continued emphasis on the use of hospital services may be due to the small number of children infected with HIV, most of whom lived in the London metropolitan area where specialist care was concentrated in a few centres. A shift from an inpatient- to an outpatient-based service was observed over time; the advent of the use of combination antiretroviral therapy in this population may further facilitate a shift in service provision and promote shared care between specialist centres, local hospital and community-based services.

HIV testing in gay sex clubs.

The purpose of this study was to evaluate a programme of human immunodeficiency virus (HIV) antibody testing at gay sex clubs. Conducting secondary analyses with 2 datasets, we evaluated HIV-testing preferences of patrons at 2 sex clubs and compared their risks to testers at a standard testing clinic. Sex club testers had significantly more partners and were significantly older than their clinic peers. Sixteen per cent of sex club testers reported that they would not test if testing were not available at the sex club. Gay sex clubs offer an opportunity to reach men at high risk for HIV, some who otherwise may not test.