Genomic Evidence of In-Flight Transmission of SARS-CoV-2 Despite Predeparture Testing.

Since the first wave of coronavirus disease in March 2020, citizens and permanent residents returning to New Zealand have been required to undergo managed isolation and quarantine (MIQ) for 14 days and mandatory testing for severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). As of October 20, 2020, of 62,698 arrivals, testing of persons in MIQ had identified 215 cases of SARS-CoV-2 infection. Among 86 passengers on a flight from Dubai, United Arab Emirates, that arrived in New Zealand on September 29, test results were positive for 7 persons in MIQ. These passengers originated from 5 different countries before a layover in Dubai; 5 had negative predeparture SARS-CoV-2 test results. To assess possible points of infection, we analyzed information about their journeys, disease progression, and virus genomic data. All 7 SARS-CoV-2 genomes were genetically identical, except for a single mutation in 1 sample. Despite predeparture testing, multiple instances of in-flight SARS-CoV-2 transmission are likely.

Dried blood spot and mini-tube blood sample collection kits for postal HIV testing services: a comparative review of successes in a real-world setting.

OBJECTIVES: This is a comparative review between using dried blood spot (DBS) and mini-tube (MT) HIV sampling kits as part of an online sexually transmitted infection (STI) postal testing service. England has recently seen increases in internet-based and postal (eHealth) STI services. Expanding accessibility and testing for patients, cost implications and narrowing the HIV undiagnosed margin are drivers for this. METHODS: In 2017, data were reviewed from an online postal STI kit requesting service at a time of transitioning from MT to DBS. We compared the STI postal kit and HIV blood sample return rates, and the successful processing/analysis rates of the DBS and MT kits. Descriptive statistics were applied to participant characteristics, with Pearson's χ2 or Fisher exact test used to demonstrate statistical differences. We also describe and calculate a 'request-to-result ratio' (RRR) for both kit types. The RRR is defined as the number of online kit requests required to produce one successfully analysed result. RESULTS: 550 STI postal kit requests from a North-West of England region were reviewed from 13 June 2017 to 22 September 2017 (275 MT, 275 DBS). Baseline characteristics between the two groups were comparable (63% woman, 90% white British and 86% heterosexual with a median age of 26 years). The successful processing rate for the DBS was 98.8% c.f. 55.7% for the MT (p<0.001). The RRR for MT was 2.96, c.f. 1.70 for DBS. There was a 5.4% false positive HIV rate in the MT c.f. none in the DBS. CONCLUSIONS: This comparative analysis suggests that in this community setting, the use of postal HIV DBS kits resulted in a significantly improved RRR compared with MT. The biggest factor was the large number of MT samples not analysed due to inadequate blood volumes. The unexpected level of false positive results in the MT samples needs confirming in larger studies.

An association between K65R and HIV-1 subtype C viruses in patients treated with multiple NRTIs.

Objectives: HIV-1 subtype C might have a greater propensity to develop K65R mutations in patients with virological failure compared with other subtypes. However, the strong association between viral subtype and confounding factors such as exposure groups and ethnicity affects the calculation of this propensity. We exploited the diversity of viral subtypes within the UK to undertake a direct comparative analysis. Patients and methods: We analysed only sequences with major IAS-defined mutations from patients with virological failure. Prevalence of K65R was related to subtype and exposure to the NRTIs that primarily select for this mutation (tenofovir, abacavir, didanosine and stavudine). A multivariate logistic regression model quantified the effect of subtype on the prevalence of K65R, adjusting for previous and current exposure to all four specified drugs. Results: Subtype B patients ( n = 3410) were mostly MSM (78%) and those with subtype C ( n = 810) were mostly heterosexual (82%). K65R was detected in 7.8% of subtype B patients compared with 14.2% of subtype C patients. The subtype difference in K65R prevalence was observed irrespective of NRTI exposure and K65R was frequently selected by abacavir, didanosine and stavudine in patients with no previous exposure to tenofovir. Multivariate logistic regression confirmed that K65R was significantly more common in subtype C viruses (adjusted OR = 2.02, 95% CI = 1.55-2.62, P < 0.001). Conclusions: Patients with subtype C HIV-1 have approximately double the frequency of K65R in our database compared with other subtypes. The exact clinical implications of this finding need to be further elucidated.

No Evidence That HIV-1 Subtype C Infection Compromises the Efficacy of Tenofovir-Containing Regimens: Cohort Study in the United Kingdom.

Concern has been expressed that tenofovir-containing regimens may have reduced effectiveness in the treatment of human immunodeficiency virus type 1 (HIV-1) subtype C infections because of a propensity for these viruses to develop a key tenofovir-associated resistance mutation. We evaluated whether subtype influenced rates of virological failure in a cohort of 8746 patients from the United Kingdom who received a standard tenofovir-containing first-line regimen and were followed for a median of 3.3 years. In unadjusted analyses, the rate of failure was approximately 2-fold higher among patients infected with subtype C virus as compared to those with subtype B virus (hazard ratio [HR], 1.86; 95% confidence interval [CI], 1.50-2.31; P < .001). However, the increased risk was greatly attenuated in analyses adjusting for demographic and clinical factors (adjusted HR, 1.14; 95% CI, .83-1.58; P = .41). There were no differences between subtypes C and subtypes non-B and non-C in either univariate or multivariate analysis. These observations imply there is no intrinsic effect of viral subtype on the efficacy of tenofovir-containing regimens.

Integrase inhibitor (INI) genotypic resistance in treatment-naive and raltegravir-experienced patients infected with diverse HIV-1 clades.

OBJECTIVES: The aim of this study was to characterize the prevalence and patterns of genotypic integrase inhibitor (INI) resistance in relation to HIV-1 clade. METHODS: The cohort comprised 533 INI-naive subjects and 255 raltegravir recipients with viraemia who underwent integrase sequencing in routine care across Europe, including 134/533 (25.1%) and 46/255 (18.0%), respectively, with non-B clades (A, C, D, F, G, CRF01, CRF02, other CRFs, complex). RESULTS: No major INI resistance-associated mutations (RAMs) occurred in INI-naive subjects. Among raltegravir recipients with viraemia (median 3523 HIV-1 RNA copies/mL), 113/255 (44.3%) had one or more major INI RAMs, most commonly N155H (45/255, 17.6%), Q148H/R/K + G140S/A (35/255, 13.7%) and Y143R/C/H (12/255, 4.7%). In addition, four (1.6%) raltegravir recipients showed novel mutations at recognized resistance sites (E92A, S147I, N155D, N155Q) and novel mutations at other integrase positions that were statistically associated with raltegravir exposure (K159Q/R, I161L/M/T/V, E170A/G). Comparing subtype B with non-B clades, Q148H/R/K occurred in 42/209 (20.1%) versus 2/46 (4.3%) subjects (P = 0.009) and G140S/A occurred in 36/209 (17.2%) versus 1/46 (2.2%) subjects (P = 0.005). Intermediate- to high-level cross-resistance to twice-daily dolutegravir was predicted in 40/255 (15.7%) subjects, more commonly in subtype B versus non-B clades (39/209, 18.7% versus 1/46, 2.2%; P = 0.003). A glycine (G) to serine (S) substitution at integrase position 140 required one nucleotide change in subtype B and two nucleotide changes in all non-B clades. CONCLUSIONS: No major INI resistance mutations occurred in INI-naive subjects. Reduced occurrence of Q148H/R/K + G140S/A was seen in non-B clades versus subtype B, and was explained by the higher genetic barrier to the G140S mutation observed in all non-B clades analysed.

Indigenous hepatitis E in England and wales from 2003 to 2012: evidence of an emerging novel phylotype of viruses.

BACKGROUND: Enhanced surveillance and molecular characterisation studies of hepatitis E virus (HEV) in England and Wales have been undertaken since 2003. The dynamics of hepatitis E have changed recently with an increase in the number of indigenous cases and an observed viral shift. METHODS: HEV antibody and RNA data were analysed to ascertain the annual number of acute infections, the HEV genotype disposition and viral phylogeny. These data were investigated in the context of collected travel history and demographic data. RESULTS: In total, 2713 acute hepatitis E cases were diagnosed, of which 1376 were indigenous infections. Travel associated cases remained steady and mainly associated with Genotype 1 infections. In contrast, major fluctuations were noted in indigenously-acquired cases with a dramatic year on year increase during 2010-2012. Molecular characterisation demonstrated indigenous infections to cluster into two distinct phylogenetic groups with the emergence of a novel group of Genotype 3 viruses coinciding with the recent increase in cases. CONCLUSIONS: HEV infection rates are dynamic in England and Wales, influenced by changing trends in indigenously-acquired cases. The recent increase in indigenous cases and the emergence of indigenous viruses not commonly circulating prior to 2010 suggest that the risk of acquiring HEV has changed.

Antiviral resistance testing.

PURPOSE OF REVIEW: Current genotypic resistance tests fail to amplify drug-resistant minority variants when they are present below 20% of the total virus population. Next-generation sequencing (NGS) addresses this issue and is being introduced into diagnostic laboratories. This review gives an overview of the resistance tests currently used and explores the opportunities and challenges that NGS genotypic resistance tests will bring. RECENT FINDINGS: The technical challenges of NGS, such as PCR and sequence-related errors, are being addressed and various assays are currently undergoing technical validation for clinical use. Although not conclusive, the data seem to suggest that NGS will be valuable for low genetic barrier drugs and certain types of tests such as the HIV-1 tropism test. Clinical validation of the reporting and interpretation of minority variant results are essential when laboratories start reporting these results. SUMMARY: The first wave of NGS technology is being rolled out in diagnostic laboratories. Antiviral test benefits include increased sensitivity and eventually cheaper antiviral resistance tests. There is a risk that low percentage minority variants may be over interpreted. This could result in antiviral drugs, which may have been effective, being possibly denied to patients if proper clinical validation studies are not performed.

Whole genome sequencing and de novo assembly identifies Sydney-like variant noroviruses and recombinants during the winter 2012/2013 outbreak in England.

BACKGROUND: Norovirus is the commonest cause of epidemic gastroenteritis among people of all ages. Outbreaks frequently occur in hospitals and the community, costing the UK an estimated £110 m per annum. An evolutionary explanation for periodic increases in norovirus cases, despite some host-specific post immunity is currently limited to the identification of obvious recombinants. Our understanding could be significantly enhanced by full length genome sequences for large numbers of intensively sampled viruses, which would also assist control and vaccine design. Our objective is to develop rapid, high-throughput, end-to-end methods yielding complete norovirus genome sequences. We apply these methods to recent English outbreaks, placing them in the wider context of the international norovirus epidemic of winter 2012. METHOD: Norovirus sequences were generated from 28 unique clinical samples by Illumina RNA sequencing (RNA-Seq) of total faecal RNA. A range of de novo sequence assemblers were attempted. The best assembler was identified by validation against three replicate samples and two norovirus qPCR negative samples, together with an additional 20 sequences determined by PCR and fractional capillary sequencing. Phylogenetic methods were used to reconstruct evolutionary relationships from the whole genome sequences. RESULTS: Full length norovirus genomes were generated from 23/28 samples. 5/28 partial norovirus genomes were associated with low viral copy numbers. The de novo assembled sequences differed from sequences determined by capillary sequencing by <0.003%. Intra-host nucleotide sequence diversity was rare, but detectable by mapping short sequence reads onto its de novo assembled consensus. Genomes similar to the Sydney 2012 strain caused 78% (18/23) of cases, consistent with its previously documented association with the winter 2012 global outbreak. Interestingly, phylogenetic analysis and recombination detection analysis of the consensus sequences identified two related viruses as recombinants, containing sequences in prior circulation to Sydney 2012 in open reading frame (ORF) 2. CONCLUSION: Our approach facilitates the rapid determination of complete norovirus genomes. This method provides high resolution of full norovirus genomes which, when coupled with detailed epidemiology, may improve the understanding of evolution and control of this important healthcare-associated pathogen.

A clinical review of mpox for the Aotearoa New Zealand clinician.

The World Health Organization declared mpox (formerly monkeypox) a Public Health Emergency of International Concern in July 2022. Aotearoa New Zealand has reported cases of mpox since July, with reports of locally acquired cases since October 2022. The 2022 global mpox outbreak highlights many features of the illness not previously described, including at-risk populations, mode of transmission, atypical clinical features, and complications. It is important that all clinicians are familiar with the variety of clinical manifestations, as patients may present to different healthcare providers, and taking lessons from the HIV pandemic, that all patients are managed without stigma or discrimination. There have been numerous publications since the outbreak began. Our narrative clinical review attempts to bring together the current clinical evidence for the New Zealand clinician.

Genomic epidemiology of syphilis in England: a population-based study.

BACKGROUND: Syphilis is a sexually transmitted bacterial infection caused by Treponema pallidum subspecies pallidum. Since 2012, syphilis rates have risen dramatically in many high-income countries, including England. Although this increase in syphilis prevalence is known to be associated with high-risk sexual activity in gay, bisexual, and other men who have sex with men (GBMSM), cases are rising in heterosexual men and women. The transmission dynamics within and between sexual networks of GBMSM and heterosexual people are not well understood. We aimed to investigate if whole genome sequencing could be used to supplement or enhance epidemiological insights around syphilis transmission. METHODS: We linked national patient demographic, geospatial, and behavioural metadata to whole T pallidum genome sequences previously generated from patient samples collected from across England between Jan 1, 2012, and Oct 31, 2018, and performed detailed phylogenomic analyses. FINDINGS: Of 497 English samples submitted for sequencing, we recovered 240 genomes (198 from the UK Health Security Agency reference laboratory and 42 from other laboratories). Three duplicate samples (same patient and collection date) were included in the main phylogenies, but removed from further analyses of English populations, leaving 237 genomes. 220 (92·8%) of 237 samples were from men, nine (3·8%) were from women, and eight (3·4%) were of unknown gender. Samples were mostly from London (n=118 [49·8%]), followed by southeast England (n=29 [12·2%]), northeast England (n=24 [10·1%]), and southwest England (n=15 [6·3%]). 180 (76·0%) of 237 genomes came from GBMSM, compared with 25 (10·5%) from those identifying as men who have sex with women, 15 (6·3%) from men with unrecorded sexual orientation, nine (3·8%) from those identifying as women who have sex with men, and eight (3·4%) from people of unknown gender and sexual orientation. Phylogenomic analysis and clustering revealed two dominant T pallidum sublineages in England. Sublineage 1 was found throughout England and across all patient groups, whereas sublineage 14 occurred predominantly in GBMSM older than 34 years and was absent from samples sequenced from the north of England. These different spatiotemporal trends, linked to demography or behaviour in the dominant sublineages, suggest they represent different sexual networks. By focusing on different regions of England we were able to distinguish a local heterosexual transmission cluster from a background of transmission in GBMSM. INTERPRETATION: These findings show that, despite extremely close genetic relationships between T pallidum genomes globally, genomics can still be used to identify putative transmission clusters for epidemiological follow-up. This could be of value for deconvoluting putative outbreaks and for informing public health interventions. FUNDING: Wellcome funding to the Sanger Institute, UK Research and Innovation, National Institute for Health and Care Research, European and Developing Countries Clinical Trials Partnership, and UK Health Security Agency.

HIV-1 superinfection.

PURPOSE OF REVIEW: This review describes the nature and frequency of HIV-1 superinfection and provides advice regarding counselling of patients in accordance with national guidelines. RECENT FINDINGS: Recent studies have demonstrated conflicting results, from no superinfection to an incidence of over 18%. We discuss the difficulties comparing studies due to population and methodological differences. SUMMARY: HIV-infected individuals should be counselled that there is risk of superinfection at all stages of HIV, but this is unlikely to be clinically significant unless transmission of resistance occurs. The risk may be as high as the risk of new incident infection in the presence of on-going exposure.

Lopinavir/ritonavir single agent therapy as a universal combination antiretroviral therapy stopping strategy: results from the STOP 1 and STOP 2 studies.

OBJECTIVES: We designed two different studies to evaluate two different combination antiretroviral therapy (cART) stopping strategies namely a 'staggered stop' approach (STOP 1 study) and a 'protected stop' approach (STOP 2 study) to find the best 'universal stop' strategy. PATIENTS AND METHODS: Patients who stopped cART for any reason were recruited. In STOP 1, 10 patients on efavirenz continued dual nucleos(t)ide reverse transcriptase inhibitors (NRTIs) for 1 week after discontinuing efavirenz. Efavirenz concentrations were measured weekly for up to 3 weeks. In STOP 2, 20 patients stopped their cART and replaced it with two tablets of lopinavir/ritonavir (Kaletra) (100/50 mg) twice daily for 4 weeks. Lopinavir, efavirenz, nevirapine and tenofovir concentrations were measured weekly for up to 4 weeks. Virological and resistance testing were performed. RESULTS: In STOP 1 five patients still had efavirenz present (median t(1/2)=148.4 h) 3 weeks after stopping. In STOP 2, 15/20 patients had a viral load (VL) of <40 copies/mL and 3/20 patients had a reduction in VL by 4 weeks. Six patients opted not to stop lopinavir/ritonavir and still had <40 copies/mL at week 8. Week 1-4 median trough lopinavir concentrations were well above the EC(95). Six patients still had detectable concentrations of original cART persisting for >1 week after stopping. No patients developed new resistance mutations. CONCLUSIONS: Plasma efavirenz concentrations can persist up to 3 weeks after patients stop efavirenz-containing regimens. This suggests a strategy of stopping efavirenz only 1 week before NRTIs may not be long enough for some individuals. The use of lopinavir/ritonavir monotherapy for a 4 week period may be an alternative pharmacologically and virologically effective universal stopping strategy which warrants further investigation.

A comparison of the performance of saliva and nasopharyngeal nucleic acid amplification testing for the detection of SARS-CoV-2 in New Zealand.

AIM: To compare detection of SARS-CoV-2 from paired nasopharyngeal swabs (NPS) and saliva using molecular methods in common use for testing swabs in New Zealand. METHOD: Samples from individuals testing positive for SARS-CoV-2 in Auckland, Wellington and Dunedin were tested at the local laboratories using methods previously established for these sample types. RESULTS: One hundred and ninety-six paired samples from unique individuals were tested, with 46 (23%) positive from either sample type, of which 43/46 (93%) tested positive from NPS, and 42/46 (91%) from saliva, indicating no significant difference in performance between sample types (p=0.69). The average Δ Ct between saliva and nasopharyngeal swabs overall across the sample set was 0.22 cycles, indicating excellent concordance; however, the difference between NPS and saliva collected from the same individual was quite variable with up to 19 cycles difference between the sample types. CONCLUSION: We found that saliva is an equivalent sample type to nasopharyngeal swab for the detection of SARS-CoV-2 in our laboratories using multiple assay combinations and is suitable for use as a diagnostic and surveillance test for selected groups of individuals.

Sensitivity and potential utility of SARS-CoV-2 rapid antigen and nucleic acid amplification tests in the context of an elimination approach.

AIM: To assess the sensitivity and potential utility of five RATs and the IDNow, Liat and Oxsed nucleic acid amplification tests (NAATs) in our population. METHOD: 39 retrospective and contrived SARS-CoV-2 positive samples were tested in parallel by standard RT-PCR and RAT. A second group of 44 samples was tested by standard RT-PCR, rapid RT-PCR and two isothermal NAAT assays. Limit of detection was compared at RT-PCR cycle thresholds for all assays. RESULTS: We found that the Cobas Liat RT-PCR had 100% concordance with conventional RT-PCR, whereas the sensitivity of other rapid NAAT assays was less at lower viral loads indicated by Cts >30 (p=0.042) and the RATs at Cts >25 (p<0.001). When applied to New Zealand testing scenarios, IDNow or Oxsed NAAT could miss up to 12% and RATs up to 44.3% of COVID-19 cases compared with the RT-PCR currently used at our laboratory. CONCLUSION: We found that the POC Cobas Liat, a platform that delivers a sample answer in 20 minutes, demonstrated equivalent performance to standard RT-PCR. However, the RATs and isothermal NAAT assays demonstrated reduced sensitivity, limiting their utility in New Zealand's currently very low prevalence setting.

Lack of N2-gene amplification on the Cepheid Xpert Xpress SARS-CoV-2 assay and potential novel causative mutations: A case series from Auckland, New Zealand.

We describe three cases with viral strains that demonstrate impaired N2-gene detection on the Cepheid Xpert Xpress SARS-CoV-2 assay, with two previously undescribed single nucleotide polymorphisms (SNPs): C29197T and G29227T. We propose that these SNPs are likely responsible since they are in close proximity to the previously described C29200T/C29200A SNPs, already shown to abolish N2-gene detection by the Xpert assay. Whether these SNPs abolish N2-gene detection by the Xpert assay individually or only in combination requires more work to elucidate.

SARS-CoV-2 antibodies in the Southern Region of New Zealand, 2020.

During New Zealand's first outbreak in early 2020 the Southern Region had the highest per capita SARS-CoV-2 infection rate. Polymerase chain reaction (PCR) testing was initially limited by a narrow case definition and limited laboratory capacity, and cases may have been missed. Our objectives were to evaluate the Abbott SARS-CoV-2 IgG nucleocapsid assay, alongside spike-based assays, and to determine the frequency of antibodies among PCR-confirmed and probable cases, and higher risk individuals in the Southern Region of New Zealand. Pre-pandemic sera (n=300) were used to establish assay specificity and sera from PCR-confirmed SARS-CoV-2 patients (n=78) to establish sensitivity. For prevalence analysis, all samples (n=1214) were tested on the Abbott assay, and all PCR-confirmed cases (n=78), probable cases (n=9), and higher risk individuals with 'grey-zone' (n=14) or positive results (n=11) were tested on four additional SARS-CoV-2 serological assays. The median time from infection onset to serum collection for PCR-confirmed cases was 14 weeks (range 11-17 weeks). The Abbott assay demonstrated a specificity of 99.7% (95% CI 98.2-99.99%) and a sensitivity of 76.9% (95% CI 66.0-85.7%). Spike-based assays demonstrated superior sensitivity ranging 89.7-94.9%. Nine previously undiagnosed sero-positive individuals were identified, and all had epidemiological risk factors. Spike-based assays demonstrated higher sensitivity than the Abbott IgG assay, likely due to temporal differences in antibody persistence. No unexpected SARS-CoV-2 infections were found in the Southern Region of New Zealand, supporting the elimination status of the country at the time this study was conducted.

Global phylogeny of Treponema pallidum lineages reveals recent expansion and spread of contemporary syphilis.

Syphilis, which is caused by the sexually transmitted bacterium Treponema pallidum subsp. pallidum, has an estimated 6.3 million cases worldwide per annum. In the past ten years, the incidence of syphilis has increased by more than 150% in some high-income countries, but the evolution and epidemiology of the epidemic are poorly understood. To characterize the global population structure of T. pallidum, we assembled a geographically and temporally diverse collection of 726 genomes from 626 clinical and 100 laboratory samples collected in 23 countries. We applied phylogenetic analyses and clustering, and found that the global syphilis population comprises just two deeply branching lineages, Nichols and SS14. Both lineages are currently circulating in 12 of the 23 countries sampled. We subdivided T. p. pallidum into 17 distinct sublineages to provide further phylodynamic resolution. Importantly, two Nichols sublineages have expanded clonally across 9 countries contemporaneously with SS14. Moreover, pairwise genome analyses revealed examples of isolates collected within the last 20 years from 14 different countries that had genetically identical core genomes, which might indicate frequent exchange through international transmission. It is striking that most samples collected before 1983 are phylogenetically distinct from more recently isolated sublineages. Using Bayesian temporal analysis, we detected a population bottleneck occurring during the late 1990s, followed by rapid population expansion in the 2000s that was driven by the dominant T. pallidum sublineages circulating today. This expansion may be linked to changing epidemiology, immune evasion or fitness under antimicrobial selection pressure, since many of the contemporary syphilis lineages we have characterized are resistant to macrolides.

Using HIV resistance tests in clinical practice.

Genotypic resistance testing is now a standard of care in HIV management. Although there are clear, published guidelines to recommend the appropriate use of these tests, clinicians and scientists still struggle to determine the optimal use of resistance tests given the finite budgets and time constraints under which they work. In this article we discuss some 'real-life' clinical situations and aim to provide a useful insight into when and where genotypic resistance testing can be optimally applied in the management of HIV-positive adults.

Human Herpes-8 virus copy to cell ratio: A diagnostic tool in primary effusion lymphoma.

Primary effusion lymphoma (PEL) is a serious sequel to Human Herpes Virus 8 (HHV8) infection in the immunosuppressed host. Usually requiring a cytological diagnosis, body cavity effusions are often referred for investigation for possible PEL. Although absence of HHV8 effectively refutes this, the presence of HHV8 DNA, though indicative is not diagnostic. Referred effusion and plasma samples from 10 patients with HHV8-related pleural and pericardial effusions were submitted for quantitative investigations. HHV8 DNA and human DNA from unseparated effusion extracts have been quantified allowing estimation of virus-to-cell ratios in effusion fluid. These ratios varied widely between 0.003 and 700. Five fluids had in excess of 106 HHV-8 DNA genome equivalents per ML (GEq/ML), ranging between 18 and 300 million GEq/ML. Four of these five effusions were from patients with cytologically proven PEL and had virus to cell (V:C) ratios between 100 and 700 to 1. The remaining high load effusion exhibited a ratio of 1.6 to 1 and came from a patient with extensive thoracic Kaposi's sarcoma. Five effusion fluids with low viral loads exhibited virus to cell ratios between 0.003 and 0.5. High effusion HHV8 load, though supportive of a diagnosis of PEL is less accurate than using virus to cell ratios.