Comparison of five SARS-CoV-2 rapid antigen detection tests in a hospital setting and performance of one antigen assay in routine practice: a useful tool to guide isolation precautions?

BACKGROUND: In a hospital setting, there is a need for rapid detection of severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) to guide isolation measures and targeted admission. AIM: To evaluate the diagnostic performance of five SARS-CoV-2 rapid nucleocapsid protein antigen detection (RAD) assays (Biosynex, Biotical, Orient Gene, Panbio and SD Biosensor), and describe the performance and impact of implementation of the SD Biosensor assay in an emergency department. METHODS: Sensitivity and specificity of the five RAD assays were analysed on 100 respiratory samples: 60 real-time reverse transcriptase polymerase chain reaction (rRT-PCR)-confirmed SARS-CoV-2-positive samples, 24 SARS-CoV-2 RNA-negative samples and 16 samples positive for other respiratory pathogens. The manufacturer's protocol was adapted to validate the antigen tests on transport media used for rRT-PCR in the authors' routine practice. The SD Biosensor RAD assay was implemented as a screening method for rapid diagnosis and targeted admission. FINDINGS: Sensitivity of the five RAD assays ranged from 88.9% to 100% for samples with cycle threshold values <26, and specificity ranged from 46.2% to 100%. During the implementation period, 4195 RAD tests were performed. Due to the rapid RAD result, 157 patients were transferred directly to the coronavirus disease 2019 (COVID-19) cohort ward instead of the regular ward (N=47) or the temporary COVID-19 ward (N=110). CONCLUSION: The SD Biosensor, Biotical and Panbio SARS-CoV-2 antigen tests showed acceptable overall performance, and identified the majority of contagious patients. In the context of high prevalence of SARS-CoV-2, RAD tests can be used as a rapid screening tool to guide infection prevention measures and aid targeted admission.

Travel history can make the difference.

Entamoeba histolytica infections are rare in developed countries such as Belgium. A 53-year-old female patient presented with 10 days of fever and mild persisting pain in the right hypochondriac despite 6 days of antibiotic therapy. The anamnesis further revealed that the patient was born in Colombia and visits her native country on a regular basis. An abdominal CT-scan demonstrated a large hepatic abscess of 10×8 cm. The diagnosis of Entamoeba histolytica- infection was confirmed with real-time PCR (RT-PCR) from the aspirated material of the abscess. Remarkably, a half year ago, this patient also presented to the gastro-enterology consultation with intermittent rectal bleeding, loose stools and abdominal discomfort. Rectosigmoidoscopy at that time showed sigmoiddiverticulosis and biopsies were taken. RT-PCR on this material was performed during this second episode and was positive for E. histolytica, confirming an episode of amoebic colitis a half year prior to the discovery of the liver abscess.

Simultaneous detection of human bocavirus and adenovirus by multiplex real-time PCR in a Belgian paediatric population.

Since the discovery of human bocavirus (hBoV), the virus has been detected worldwide in respiratory tract samples from young children by various polymerase chain reaction (PCR) assays and real-time PCRs (Q-PCR). Until now, no data have been reported on the presence of hBoV in Belgium and the detection of hBoV in a multiplex Q-PCR setting has not been described. The aim of this study was to develop a fast and reliable multiplex Q-PCR for the simultaneous detection of hBoV DNA and adenovirus (AdV) DNA. During the winter of 2004-2005, 445 nasopharyngeal aspirates (NPAs) were analysed from 404 Belgian children up to 5 years old with acute respiratory tract infections (ARTIs). (Co)infections with hBoV, AdV, respiratory syncytial virus (RSV), human metapneumovirus (hMPV) and influenza A virus were investigated. A viral agent was detected in 61% (n = 272/445) of the NPAs. Multiplex Q-PCR found a prevalence of 11% (n = 51/445) hBoV and 13% (n = 58/445) AdV. Coinfections were more frequently found with AdV (62%; n = 36/58) than with hBoV (49%; n = 25/51). Follow-up samples were available from 22 patients with ARTIs. In three patients, hBoV DNA persisted for one month. Multiplex Q-PCR may help in closing the diagnostic gap by addressing a broader range of potential respiratory pathogens.

Contribution of the FilmArray Respiratory Panel in the management of adult and pediatric patients attending the emergency room during 2015-2016 influenza epidemics: An interventional study.

AIM: To evaluate the contribution of a multiplex PCR for respiratory viruses on antibiotic and antiviral prescription, ancillary test prescription, admission and length of stay of patients. METHODS: Two hundred ninety-one adult and pediatric patients visiting the emergency department during the 2015-2016 influenza epidemic were prospectively included and immediately tested 24/7 using the FilmArray Respiratory Panel. The results were communicated to the practitioner in charge as soon as they became available. Clinical and biological data were gathered and analyzed. FINDINGS: Results from the FilmArray Respiratory Panel do not appear to impact admission or antibiotic prescription, with the exception of a lower admission rate for children who tested positive for influenza B. Parameters that account for the clinical decisions evaluated are CRP level, white blood cell count, suspected or proven bacterial infection and, for adult patients only, signs of respiratory distress. Length of stay is also not significantly different between patients with a positive and a negative result. A rapid influenza test result permits a more appropriate prescription of oseltamivir.

Prospective evaluation of diagnostic tools for respiratory viruses in children and adults.

AIM: To compare the performances of molecular and non-molecular tests to diagnose respiratory viral infections and to evaluate the pros and contras of each technique. METHODS: Two hundred ninety-nine respiratory samples were prospectively explored using multiplex molecular techniques (FilmArray Respiratory Panel, Clart Pneumovir), immunological techniques (direct fluorescent assay, lateral flow chromatography) and cell cultures. FINDINGS: Molecular techniques permitted the recovery of up to 50% more respiratory pathogens in comparison to non-molecular methods. FilmArray detected at least 30% more pathogens than Clart Pneumovir which could be explained by the differences in their technical designs. The turnaround time under 2 hours for the FilmArray permitted delivery of results when patients were still in the emergency room.

Pan-genome multilocus sequence typing and outbreak-specific reference-based single nucleotide polymorphism analysis to resolve two concurrent Staphylococcus aureus outbreaks in neonatal services.

We used a two-step whole genome sequencing analysis for resolving two concurrent outbreaks in two neonatal services in Belgium, caused by exfoliative toxin A-encoding-gene-positive (eta+) methicillin-susceptible Staphylococcus aureus with an otherwise sporadic spa-type t209 (ST-109). Outbreak A involved 19 neonates and one healthcare worker in a Brussels hospital from May 2011 to October 2013. After a first episode interrupted by decolonization procedures applied over 7 months, the outbreak resumed concomitantly with the onset of outbreak B in a hospital in Asse, comprising 11 neonates and one healthcare worker from mid-2012 to January 2013. Pan-genome multilocus sequence typing, defined on the basis of 42 core and accessory reference genomes, and single-nucleotide polymorphisms mapped on an outbreak-specific de novo assembly were used to compare 28 available outbreak isolates and 19 eta+/spa-type t209 isolates identified by routine or nationwide surveillance. Pan-genome multilocus sequence typing showed that the outbreaks were caused by independent clones not closely related to any of the surveillance isolates. Isolates from only ten cases with overlapping stays in outbreak A, including four pairs of twins, showed no or only a single nucleotide polymorphism variation, indicating limited sequential transmission. Detection of larger genomic variation, even from the start of the outbreak, pointed to sporadic seeding from a pre-existing exogenous source, which persisted throughout the whole course of outbreak A. Whole genome sequencing analysis can provide unique fine-tuned insights into transmission pathways of complex outbreaks even at their inception, which, with timely use, could valuably guide efforts for early source identification.

Staphylococcus saprophyticus bacteremia after ESWL in an immunocompetent woman.

Staphylococcus saprophyticus is a well-known cause of uncomplicated urinary tract infections, especially in young and sexually active women. Presence in blood cultures is rare and often attributed to contamination. When bacteremia is significant, it occurs mostly in patients with hematologic malignancies and is predominantly catheter-related. However, we describe a case of significant bacteremia with S. saprophyticus associated with urinary tract infection after extracorporeal shock wave lithotripsy of an ureterolithiasis in an otherwise healthy patient.

Simple algorithm derived from a geno-/phenotypic database to predict HIV-1 protease inhibitor resistance.

BACKGROUND: Resistance against protease inhibitors (PI) can either be analysed genotypically or phenotypically. However, the interpretation of genotypic data is difficult, particularly for PI, because of the unknown contributions of several mutations to resistance and cross-resistance. OBJECTIVE: Development of an algorithm to predict PI phenotype from genotypic data. METHODS: Recombinant viruses containing patient-derived protease genes were analysed for sensitivity to indinavir, saquinavir, ritonavir and nelfinavir. Drug resistance-associated mutations were determined by direct sequencing. geno- and phenotypic data were compared for 119 samples from 97 HIV-1 infected patients. RESULTS: Samples with one or two mutations in the gene for the protease were phenotypically sensitive in 74.3%, whereas 83.6% of samples with five or more mutations were resistant against all PI tested. Some mutations (361, 63P, 71V/T, 771) were frequent both in sensitive and resistant samples, whereas others (241, 30N, 461/L, 48V, 54V, 82A/F/T/S, 84V, 90M) were predominantly present in resistant samples. Therefore, the presence or absence of a single drug resistance-associated mutation predicted phenotypic PI resistance with high sensitivity (96.5-100%) but low specificity (13.3-57.4%). A more specific algorithm was obtained by taking into account the total number of drug resistance-associated mutations in the gene for the protease and restricting these to certain key positions for the PI. The algorithm was subsequently validated by analysis of 72 independent samples. CONCLUSION: With an optimized algorithm, phenotypic PI resistance can be predicted by viral genotype with good sensitivity (89.1-93.0%) and specificity (82.6-93.3%). The reliability and relevance of this algorithm should be further evaluated in clinical practice.

Prevalence of genotypic resistance among antiretroviral drug-naive HIV-1-infected patients in Belgium.

OBJECTIVES: To estimate the prevalence and the evolution over time (1995-1998) of genotypic resistance to antiviral drugs in antiretroviral drug-naive HIV-1-infected patients in Belgium. DESIGN: Belgian Aids Reference Laboratories provided retrospective samples and clinical data from antiretroviral drug-naive HIV-1-infected patients who visited the hospital for the first time in 1995 (n=45), 1997 (n=75) and 1998 (n=111). Genotypic resistance to the three available classes of drugs was monitored using the Line Probe Assay (Innogenetics, Gent, Belgium). Additionally, ARMS-151 was performed for scoring multinucleoside resistance. RESULTS: The prevalence of genotypic resistance at baseline to nucleoside analogue reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were each between 10% and 20% for 1995, 1997 and 1998 without an increasing trend over time. For NRTIs, resistance mutations were mainly related to zidovudine in 1995, whereas in 1997 and 1998 baseline resistance was scored for zidovudine, lamivudine or for both drugs simultaneously. No patients displayed the multi-nucleoside resistance Q151M mutation. Baseline resistance mutations to protease inhibitors (PIs) did not rise significantly: 4.4% in 1995, 8% in 1997 and 9.9% in 1998. When scoring any resistance-related mutation, 26.6% displayed genotypic baseline resistance in 1995, 26.6% in 1997 and 31.5% in 1998. DISCUSSION: The prevalence of genotypic baseline resistance to any drug, as scored with LiPA, in naive HIV-1 patients in Belgium is 29%, with baseline resistance mutations to one or several drugs from all available classes of antiviral drugs. The ability of LiPA to pick up minor variants could be an explanation for the higher overall prevalence we observe, when compared to recent estimates in other countries of 16.3% and 22%, which were based on sequencing methods. According to the European guidelines for resistance testing, resistance testing in Belgium before starting antiviral therapy should be considered.

Baseline HIV type 1 genotypic resistance to a newly added nucleoside analog is predictive of virologic failure of the new therapy.

We evaluated the predictive value of baseline HIV-1 genotypic resistance mutations for failure of a nucleoside reverse transcriptase inhibitor (NRTI) containing therapy. The change in therapy of 88 HIV-1-infected patients was analyzed retrospectively, relating the genotypic resistance profile at baseline to the evolution of viral load and CD4+ T cell counts. Genotypic resistance at baseline and at 6 months was evaluated with the LiPA HIV-1 RT, which detects mutations at codons 41, 69, 70, 74, 184, and 215. At 1 to 3 months after change in therapy, patients without preexisting resistance mutations to the new drug (group S) had a significantly better evolution in viral load (reduction of 0.37 log(10)) compared with patients with known preexisting resistance mutation(s) (group R) (increase of 0.08 log(10)). This difference was particularly striking for patients with the baseline M184V mutation and whose treatment was modified by the addition of lamivudine. After 6 months the median difference in viral load evolution between the two groups increased to 0.61 log(10): the viral load of patients of group S was still 0.18 log(10) below baseline while patients of group R had an increase of 0.43 log(10) in viral load above baseline. Changes in CD4+ T cell counts were not significantly different. The evolution in viral load in HIV-1-infected patients with and without baseline resistance mutation(s) toward a newly added NRTI is significantly different at 1-3 months and at 6 months after changing or adding one NRTI.

Initiation of HAART in drug-naive HIV type 1 patients prevents viral breakthrough for a median period of 35.5 months in 60% of the patients.

The introduction of potent combinations of antiviral drugs is a major breakthrough in the treatment of HIV. We investigated the long-term virologic outcome and the development of resistance after initiating highly active antiretroviral therapy (HAART) in drug-naive patients in daily clinical practice. Twenty-five treatment-naive HIV-1 patients were started on HAART. Fifteen patients responded with a drop in viral load below the limit of detection during 35.5 (interquartile range: 7) months of therapy. In 6 of 10 patients with virologic failure, virus with resistance-related mutations against the received drugs emerged. Compared with responders (R), nonresponding (NR) patients were in a later disease stage at therapy start (p = 0.0089) with lower CD4 cell counts at baseline (p = 0.040), and a lower proportion of nonresponders showed protease inhibitor (PI) levels above C(min) (p = 0.049). More NR patients showed secondary PI mutations at baseline (p = 0.079), and the CCR2-64I coreceptor polymorphism was absent among NR patients, compared with 38.5% of R patients displaying CCR2-64I (p = 0.053), although the differences were not significant. In conclusion, starting HAART in antiretroviral drug-naive HIV-infected patients followed in daily clinical practice prevented viral breakthrough for up to 44 months in 60% of the patients. Virologic failure was associated with the development of resistance-related mutations, a later stage of disease at start of therapy and lower PI drug levels.

Anti-human immunodeficiency virus drug combination strategies.

It is now generally accepted that mono- and bitherapy for human immunodeficiency virus type 1 (HIV-1) infection are only transiently efficient mainly due to virus drug resistance. To obtain a sustained benefit from antiviral therapy, current guidelines recommend at least triple-drug combinations, or the so-called highly active antiretroviral therapy (HAART). In some patients, HAART can be problematic, either because it is difficult to remain compliant or because previous suboptimum therapies have limited the choice of drugs. For compliant drug-naive patients, HAART should be able to offer long-term virus suppression, when changing from first- to second- to third-line HAART at drug failure. Long-term treatment might ultimately result in multi-drug resistant virus leaving few options for salvage therapy. HIV drug resistance testing to guide this salvage therapy and the development of new drugs to allow new options will therefore remain priorities in anti-HIV drug research.

Study of the impact of HIV genotypic drug resistance testing on therapy efficacy.

During recent years significant progress has been made in the treatment of HIV-1, at least in part due to the availability of potent antiretroviral drugs. The goal of the current treatment strategies is to inhibit the viral replication as completely as possible by using a combination of 3 or more antiretroviral drugs. This Highly Active Antiretroviral Therapy (HAART) has radically changed the clinical outcome of HIV, leading to decreased mortality and morbidity, at least in developed countries. Additionally to the advent of new and potent drugs, demonstrations of the prognostic value of the CD4 cell count and the plasma viral load were of major importance in the development of therapeutic strategies. Especially the ability of viral load assays to assess accurately the true level of viral replication, led to a better understanding of the pathogenesis of the disease. HIV proved to be a highly dynamic infection even during the period of clinical latency. The initial enthusiasm that HAART could radically change the outcome of HIV was cooled off in face of the difficulties in real life associated with the complex treatment strategies. Besides long-term side effects and suboptimal drug potency, the emergence of resistant virus and the necessity of perfect therapy adherence are major concerns for obtaining a sustained control of viral replication. In this study we focused on HIV resistance, which remains one of the major threats for a sustained response to antiretroviral therapy. HIV proved to be able to develop resistance to all currently used antiretroviral drugs. The high replication rate of the virus together with the low fidelity of the viral reverse transcriptase, from the basis for the presence of enormous amounts of viral variants. Whenever viral replication is ongoing in the presence of antiretroviral drugs, these variants that escape the inhibitory effects of the drugs will be selected. Although the knowledge in the field of HIV resistance has expanded enormously, many issues need to be answered. Genotypic and phenotypic resistance patterns are evolving continuously, due to changes in the treatment strategies. Moreover the relation between drug resistance and therapy failure needs further investigation, in order to prove the relevance of performing resistance testing in the follow-up of HIV-infected patients. The wide availability of antiretroviral drugs has led to the transmission of resistant HIV. Infection with HIV resistant to one or more antiretroviral drugs has been observed to occur through the different transmission routes. In a first study we assessed the prevalence of genotypic resistance to antiretroviral drugs in Belgian antiretroviral-naïve HIV-infected patients. We observed that HIV strains with resistance-related mutations to one or more classes of antiretroviral drugs are not uncommon in the Belgian naïve patients. Furthermore the inclusion of samples from patients visiting the Belgian hospitals for the first time in 1995, 1997 and 1998, showed that the overall prevalence of baseline genotypic resistance remains rather constant (26-30%). The increasing trend in genotypic baseline resistance to 3TC (2% to 6.3%) and PIs (4.4% to 9.9%) as well as the decrease in ZDV-resistance (13.3% to 5.4%), reflect the change in treatment strategies, and resistance to these drugs is most probably caused by transmission of variants with resistance mutations selected during therapy. The presence of NNRTI-related mutations (around 16%) is likely to reflect the occurrence of baseline polymorphisms, since NNRTI-related mutations can occur without a replication deficit for the virus. Moreover, despite the rather recent introduction of NNRTIs into the clinic from 1997 onwards, no clear trend in NNRTI baseline resistance over time is observed. The best current therapeutic strategy for HIV-infected patients is to start antiretroviral therapy with HAART in order to avoid the accumulation of resistance towards drugs in less suppressive regimens. In a second study, we showed that the start of HAART in antiretroviral-naive HIV-patients in daily clinical practice could prevent viral breakthrough for up to 44 months in 60% of patients (n = 25). Six of 10 patients with virologic failure developed resistance to the drugs included in their treatment regimens. In comparison to patients with a sustained virologic response, patients with virologic failure were in a later disease stage when starting therapy and showed lower PI drug-levels, what can be an indication of poor adherence. Despite a poor virologic response for some of them, a rise in CD4 cell count was observed for all patients during the study period. A large number of HIV-infected patients started treatment in the pre-HAART period. The use of NRTIs is mono- or bitherapy was not able to prevent the development of resistant virus. In a third study we studied the prevalence and characteristics of 2 patterns of multinucleoside resistance (MNR) in European patients (n = 755). In patients without NRTI-exposure or with exposure to only one NRTI, no MNR was observed. MNR was present in low prevalence (each pattern < 2%) in patients pretreated with multiple NRTIs. Despite this low prevalence, MNR should be closely monitored, since it results in broad cross-resistance to NRTIs in vitro and a poor therapy response in vivo. We also assessed the predictive value of baseline resistance on the virologic response to later added drugs. The genotype of patients starting or changing a therapy consisting solely of NRTIs was analyzed at baseline and 6 months later. In patients without genotypic mutations towards the added drug the virologic response was significantly better compared to patients with baseline resistance. At 6 months however, both patient groups showed a rise in viral load due to the accumulation of NRTI-related mutations under the presence of poorly suppressive regimens, although the difference between the two groups remained significant. In this study, and also in studies reported by others, the presence of baseline resistance has a high predictive value for therapy failure, while the absence of resistance is not predictive for therapy response. Suboptimal adherence may be one of the reasons of the poor predictive value of the absence of baseline resistance for a good therapy response. In a last observational study, we investigated the relation between adherence, the presence and development of genotypic resistance and the virologic response in patients during HAART therapy. Adherence to 1 protease inhibitor was monitored using Electronic Event Monitoring. Patients with perfect therapy adherence and in particular without drug holidays, can control viral replication provided that the activity of the drugs included in the combination is not entirely compromised by the presence of baseline resistance mutations. In our patient population, reduced adherence resulted in therapy failure, mostly associated with a subsequent accumulation of resistance mutations. In conclusion, the outcome of the HIV disease has been revolutionarily changed with the advent of HAART. Both resistance and treatment adherence are crucial factors in determining the therapy response. Retrospective studies, such as ours, and a limited number of prospective trials already proved the short-term benefit of therapy switch based on the results of resistance tests in addition to standard of care. To ultimately define the role of tools as resistance testing and adherence monitoring with eventual adherence interventions, more prospective trials are needed as well in treatment-naïve as in experienced patients.

An isocenter position verification device for electronic portal imaging: physical and dosimetrical characteristics.

The physical and dosimetrical characteristics of a device, designed to visualize the isocenter position on electronic portal images, were examined. The device, to be mounted on the gantry head of the accelerator, containing five spheric lead markers, was designed in order to visualize the isocenter position on portal images. A quality control device was designed to check the reliability of this technique. The disturbance of the dose distribution by the markers was studied with gel dosimetry. The use of markers resulted in a precise and accurate method to visualize the isocenter on portal images. A maximum underdosage of 11%, due to attenuation by the markers, was observed. The use of markers to visualize the isocenter position on portal images, is a fast and reliable method when analyzing patient setup errors with online electronic portal imaging.

Let's not forget herpes simplex virus in case of fulminant hepatic failure.

Fulminant herpes simplex virus (HSV) hepatitis is a rare condition, which is usually identified only after orthotopic liver transplantation (OLT) or at autopsy. The most commonly affected individuals are immunosuppressed patients, although HSV hepatitis can occur in immunocompetent patients as well. A high degree of suspicion combined with early diagnostic modalities may improve survival. We present a case report of fulminant herpetic hepatitis, requiring OLT. In addition, a review of the literature was performed.

Brain abscesses with Peptostreptococcus: not unusual after oesophageal dilatation.

A case of a brain abscess following oesophageal dilatation for caustic stenosis in a 67-year old woman is reported. Previously reported cases of brain abscess development after oesophageal dilatation are reviewed. Following oesophageal dilatation, bacteraemia and fever are common but the occurrence of metastatic brain abscesses is rare. The clinical presentation is non-specific, with high fever and neurological findings as most reported signs. The isolated organisms belong to the normal oropharyngeal bacterial flora. Prognosis is satisfactory after early diagnosis and correct management. As a result, clinicians dealing with oesophageal strictures should keep in mind that brain abscess formation is a potential complication of oesophageal dilatation.

Atypical pneumonia due to Chlamydophila psittaci: 3 case reports and review of literature.

Chlamydophila psittaci is the causative agent of psittacosis or ornithosis. The disease is transmitted to men predominantly from birds. Most commonly noted symptoms are fever, headache and cough, but a number of other symptoms or complications may arise such as renal impairment, hepatitis or neurological symptoms. In this article 3 cases of psittacosis are presented, with a review of the literature with emphasis on laboratory diagnosis.

Monitoring of herpes simplex virus in the lower respiratory tract of critically ill patients using real-time PCR: a prospective study.

Herpes simplex virus (HSV) has increasingly been associated with pulmonary disease in critically ill patients. However, the clinical relevance of HSV is still a topic of debate. Monitoring of HSV in a quantitative way could potentially give relevant information on its role in the pathogenesis of lower respiratory tract infection. A fast and reliable quantitative real-time PCR (Q-PCR) for the quantitative detection of HSV-1 and HSV-2 DNA was developed. A prospective observational study was performed in an intensive-care unit (ICU) to monitor the HSV viral load in lower respiratory tract aspirates of long-term mechanically ventilated patients. HSV was common in the lower respiratory tract (LRT) of critically ill patients with mechanical ventilation for at least 48 h (62%, n = 65/105). Detection of HSV was significantly associated with prolonged mechanical ventilation (p <0.01), prolonged ICU stay (p <0.01), and development of ventilator-associated pneumonia (p = 0.02). Corticosteroid administration (p <0.01) in the ICU and anti-HSV IgG seropositivity (p <0.01) were risk factors for the occurrence of HSV in the LRT. The fact that no HSV-seronegative patient became positive suggests that all HSV DNA-positive patients had HSV reactivations. Monitoring the HSV viral load in the LRT of critically ill patients showed a typical homogeneous pattern of HSV kinetics. HSV emerged in tracheal and bronchial aspirates after a median of 7 days of intubation (5-11 days), and this was followed by an exponential increase (c. 1 log copies/mL/day) to reach very high HSV peaks (10(6)-10(10) copies/mL) in 78% of the HSV DNA-positive patients.

Evaluation of two commercial kits for the detection of genotypic drug resistance on a panel of HIV type 1 subtypes A through J.

We compared the two commercially available sequencing kits for HIV-1 drug resistance testing, the ViroSeq Genotyping System (Applied Biosystems, Foster City, CA, U.S.A.) and the TRUGENE HIV-1 Genotyping Kit (Visible Genetics, Inc., Toronto, Ontario, Canada), with our in-house genotyping system. Fifteen viral isolates from African patients (6 treated and 9 untreated) covering a panel of HIV-1 subtypes A through J and 7 plasma samples from Belgian and African patients (2 treated and 5 untreated) were tested. All the samples could be amplified and sequenced by the three systems; however, for all systems, alternative amplification/sequencing primers had to be used for some samples belonging to subtype B as well as to other subtypes. The consensus sequence was partially derived from only one strand for the in-house system and for the ViroSeq Genotyping System. The TRUGENE HIV-1 Genotyping Kit scored the highest number of ambiguities, followed by the ViroSeq Genotyping System and the in-house system. For 11 samples, these differences in reporting mixtures affected 14 resistance-related positions, which altered the interpretation toward protease inhibitors for 2 samples when using version 1.2 RetroGram software (Virology Networks, Utrecht, The Netherlands). All three systems were able to sequence diluted samples with a viral load down to 10 3 or 10 4 RNA copies/ml. Our data therefore suggest that the performance of amplification and sequencing primers must be improved to allow fast and reliable resistance testing for all HIV-1 subtypes.

Phenotypic assays and sequencing are less sensitive than point mutation assays for detection of resistance in mixed HIV-1 genotypic populations.

The sensitivity and discriminatory power of the 151 and 215 amplification refractory mutation system (ARMS) were evaluated, and their performance for the detection of drug resistance in mixed genotypic populations of the reverse transcription (RT) gene of HIV-1 were compared with T7 sequencing, cycle sequencing, the line probe assay (LiPA) HIV-1 RT test, and the recombinant virus assay (RVA). ARMS and the LiPA HIV-1 RT test were shown to be able to detect minor variants that in particular cases comprised only 1%. T7 sequencing on an ALF semiautomated sequencer could correctly score mixtures only when variants were present at 50%. Cycle sequencing on an ABI PRISM 310 improved the sensitivity for mixtures to about 25%. Using RVA, it was shown that at least 50% of the virus population needed to carry the resistance mutation at codon 184 to afford phenotypic resistance against lamivudine. The two point mutation assays therefore proved to be more sensitive methods than sequencing and RVA to reliably determine a gradual shift in HIV-1 drug resistance mutations in follow-up of patients infected with HIV-1. In 4 of 5 treated patients who were followed by ARMS, a gradual shift in resistant genotypic populations was observed during a period of 6 to 19 months. For 1 patient, a shift from wild to mutant type at position 151 occurred within 2 months, without mixed genotypic intermediate type's being detected.