Evaluation of extraction-free RT-qPCR methods for SARS-CoV-2 diagnostics.

Extraction-based real-time reverse transcription quantitative polymerase chain reaction (RT-qPCR) is currently the "gold standard" in SARS-CoV-2 diagnostics. However, some extraction-free RT-qPCR techniques have recently been developed. In this study, we compared the sensitivity of traditional extraction-based, heated extraction-free, and unheated extraction-free RT-qPCR methods for SARS-CoV-2 detection in nasopharyngeal swabs from symptomatic individuals. The unheated extraction-free method showed perfect agreement with the standard extraction-based RT-qPCR. By contrast, the heat-treated technique was associated with an 8.2% false negativity rate. Unheated extraction-free RT-qPCR for the molecular diagnosis of SARS-CoV-2 is a valuable alternative to the traditional extraction-based methods and may accelerate turnaround times by about two hours.

Rapid diagnosis of SARS-CoV-2 pneumonia on lower respiratory tract specimens.

BACKGROUND: The ongoing SARS-CoV-2 pandemic requires the availability of accurate and rapid diagnostic tests, especially in such clinical settings as emergency and intensive care units. The objective of this study was to evaluate the diagnostic performance of the Vivalytic SARS-CoV-2 rapid PCR kit in lower respiratory tract (LRT) specimens. METHODS: Consecutive LRT specimens (bronchoalveolar lavage and bronchoaspirates) were collected from Intensive Care Units of San Martino Hospital (Genoa, Italy) between November 2020 and January 2021. All samples underwent RT-PCR testing by means of the Allplex™ SARS-CoV-2 assay (Seegene Inc., South Korea). On the basis of RT-PCR results, specimens were categorized as negative, positive with high viral load [cycle threshold (Ct) ≤ 30] and positive with low viral load (Ct of 31-35). A 1:1:1 ratio was used to achieve a sample size of 75. All specimens were subsequently tested by means of the Vivalytic SARS-CoV-2 rapid PCR assay (Bosch Healthcare Solutions GmbH, Germany). The diagnostic performance of this assay was assessed against RT-PCR through the calculation of accuracy, Cohen's κ, sensitivity, specificity and expected positive (PPV) and negative (NPV) predictive values. RESULTS: The overall diagnostic accuracy of the Vivalytic SARS-CoV-2 was 97.3% (95% CI: 90.9-99.3%), with an excellent Cohen's κ of 0.94 (95% CI: 0.72-1). Sensitivity and specificity were 96% (95% CI: 86.5-98.9%) and 100% (95% CI: 86.7-100%), respectively. In samples with high viral loads, sensitivity was 100% (Table 1). The distributions of E gene Ct values were similar (Wilcoxon's test: p = 0.070), with medians of 35 (IQR: 25-36) and 35 (IQR: 25-35) on Vivalytic and RT-PCR, respectively (Fig. 1). NPV and PPV was 92.6% and 100%, respectively. Table 1 Demographic characteristics and data sample type of the study cases (N = 75) Male, N (%) 56 (74.6%) Age (yr), Median (IQR) 65 (31-81) BAS, N (%) 43 (57.3%)  Negative 30.2%  Positive-High viral load [Ct ≤ 30] 27.9%  Positive-Low viral load [Ct 31-35] 41.9% BAL, N (%) 32 (42.7%)  Negative 37.5%  Positive-High viral load [Ct ≤ 30] 40.6%  Positive-Low viral load [Ct 31-35] 21.9% Data were expressed as proportions for categorical variables. Specimens were categorized into negative, positive with high viral load [cycle threshold (Ct) ≤ 30] and positive with low viral load (Ct of 31-35). BAS bronchoaspirates, BAL bronchoalveolar lavage, Ct cycle threshold Fig. 1 Distribution of E gene cycle threshold values of the rapid PCR and RT-PCR CONCLUSIONS: Vivalytic SARS-CoV-2 can be used effectively on LRT specimens following sample liquefaction. It is a feasible and highly accurate molecular procedure, especially in samples with high viral loads. This assay yields results in about 40 min, and may therefore accelerate clinical decision-making in urgent/emergency situations.

Efficacy of lamivudine prophylaxis in preventing hepatitis B virus reactivation in patients with resolved infection undergoing allogeneic SCT and receiving rituximab.

PURPOSE: Hepatitis B virus (HBV) reactivation during immunosuppressive therapy is common in patients with hematological malignancies, even in case of resolved infection. Prophylaxis of HBV reactivation is universally recommended in stem cell transplant (SCT) recipients and patients treated with anti-CD20 agents (i.e., rituximab). Despite its well-established favorable safety profile, lamivudine (LAM) use in prophylaxis has been debated because of the possible emergence of resistant viral strains. The aim of this study was to investigate the efficacy of LAM in preventing HBV reactivation in allogeneic SCT recipients with a resolved HBV infection. METHODS: Patients who received first allogeneic SCT in years 2009-2016 were evaluated. Sixty-three patients with resolved infection received LAM prophylaxis and were included in the study. Baseline and post-SCT characteristics were recorded, including rituximab exposure, length of LAM prophylaxis, and time from transplant to the last clinical and virological follow-up. RESULTS: Overall, 39 patients (62%) were male, 39 (62%) had acute myeloid leukemia, 38 (60%) received transplant from haploidentical donor, 29 (53%) received myeloablative conditioning, and 15 (24%) received rituximab post-transplant. Median clinical follow-up was 24 months after SCT (range 0.3-97); median virological follow-up 16 months (range 0.3-78), and median length of LAM prophylaxis of 14.5 months (range 0.3-78). No patient experienced HBV reactivation while on LAM prophylaxis. One patient experienced reactivation 8 months after discontinuing prophylaxis. CONCLUSIONS: In this high-risk population, LAM prophylaxis was effective in preventing HBV reactivation in patients with resolved infection. It should be considered a reasonable first-line prophylactic agent to be administered in this setting.

Case report: management and HBV sequencing in a patient co-infected with HBV and HIV failing tenofovir.

Nucleos(t)ide analogs such as tenofovir, lamivudine, or emtricitabine are active against both HBV and HIV. Tenofovir confers potent and durable HBV-DNA suppression but the best strategy in case of resistance of HBV to tenofovir remains unknown. A case of a 22-year-old patient with co-infection with HBV and HIV transmitted perinatally is reported. After prolonged and intermittent treatment of HIV with lamivudine and tenofovir, HBV became resistant to lamivudine. Subsequently, clinical resistance to tenofovir occurred, manifesting as HBV-DNA breakthrough. The non-compliance was reasonable excluded and HIV-RNA remained constantly suppressed. Entecavir (1 mg daily) was added and the combination therapy resulted in a rapid and continuous suppression of HBV-DNA for over 12 months. The treatment was well-tolerated and safe. No known mutations, such as rtA181T/V associated with rtN236T or A194T that are associated with reduced susceptibility or resistance to tenofovir were detected. However, a unique and complex HBV substitution pattern was found: with a development of rtR192PR mutation at the time of virological failure. Adding entecavir to failing therapy with tenofovir and emtricitabine was feasible, well-tolerated and resulted in virological success. The rtR192PR, which is located in the B domain near the rtA194T, occurring in a context of a very complex substitutions patterns, might be associated with resistance to tenofovir.

Lamivudine resistance mutations in European patients with hepatitis B and patients co-infected with HIV and hepatitis B.

Evaluation of resistance pattern in patients with chronic hepatitis B. Retrospective study of hepatitis B virus (HBV) resistance mutations in patients found viraemic after first-line treatment. HBV viral load was determined by a real-time polymerase chain reaction and the substitutions in HBV-DNA were studied by polymerase sequencing test. First line treatment had failed in 12 out of 33 patients (36%) receiving anti-HBV drugs. The 12 patients with persistent viraemia were all lamivudine (LAM) experienced and 7 had a polymerase sequencing test available. LAM substitution mutations L180M + M204V/I were found in six out of seven cases, with an accompanying V173L mutation in three cases. These mutations were also related with changes in HBsAg. The use of potent drugs in the first line anti-HBV therapy may reduce the resistance mutations in the future.

Clinical and epidemiological characteristics of COVID-19 during the early phase of the SARS-CoV-2 pandemic: a cross-sectional study among medical school physicians and residents employed in a regional reference teaching hospital in Northern Italy.

OBJECTIVES: The aim of the study was to evaluate the clinical presentation and burden of SARS-CoV-2 infections among medical school physicians and residents, mainly young medical doctors. The awareness of COVID­19 clinical manifestations can improve the early detection of mild cases, possibly reducing further transmission to colleagues and patients. MATERIAL AND METHODS: The study was carried out in March-May 2020, involving medical school physicians in a teaching hospital in northern Italy, with a working population of 881 medical doctors. Data collection was performed using a structured form investigating clinical and epidemiological information. RESULTS: One hundred sixty-two medical doctors contacted the Occupational Health Service reporting acute respiratory symptoms or close contact exposure to a confirmed COVID­19 case. Among the confirmed COVID­19 cases, most were male doctors during residency, and 85% presented a mild clinical picture. Fever (70.3%) and cough (51.4%) represented the most prevalent symptoms of COVID­19. As revealed by the univariate analysis, the prevalence of real-time reverse transcriptase-polymerase chain reaction (RT-PCR) positivity increased with age (OR = 1.08, 95% CI: 1.02-1.14, p = 0.012), working in a COVID­19 ward (OR = 3.33, 95% CI: 1.09-10.21, p = 0.031), presenting alteration or loss of smell/taste (OR = 10.00, 95%CI: 2.80-35.69, p < 0.001) and myalgia (OR = 3.20, 95% CI: 1.00-10.26, p = 0.046), while being a resident (OR = 0.20, 95% CI: 0.05-0.80, p = 0.030) was associated with reduced odds of being infected, compared to staff physicians. Age and loss of smell/taste were the only factors independently associated with RT-PCR positivity. CONCLUSIONS: The majority of COVID­19 cases showed a mild clinical syndrome, ranging from absence or paucity of symptoms to common cold or influenza-like symptoms. The findings of the present study increase the accuracy of the clinical diagnosis for the prompt identification and management of suspected COVID­19 cases, being particularly useful during resurges of the SARS-CoV-2 pandemic. Int J Occup Med Environ Health. 2021;34(2):189-201.

Comparative Diagnostic Performance of a Novel Reverse Transcription Loop-Mediated Isothermal Amplification (RT-LAMP) Kit for the Rapid Detection of SARS-CoV-2.

Although the reverse transcription-polymerase chain reaction (RT-PCR) is considered a standard-of-care assay for the laboratory diagnosis of SARS-CoV-2, several limitations of this method have been described. Reverse transcription loop-mediated isothermal amplification (RT-LAMP) is an alternative molecular assay and is potentially able to overcome some intrinsic shortcomings of RT-PCR. In this study, we evaluated the diagnostic performance of the novel HG COVID-19 RT-LAMP assay. In this retrospective analysis, a total of 400 routinely collected leftover nasopharyngeal samples with a known RT-PCR result were tested by means of the HG COVID-19 RT-LAMP assay. The overall sensitivity and specificity values of HG COVID-19 RT-LAMP versus RT-PCR were 97.0% (95% CI: 93.6-98.9%) and 98.5% (95% CI: 95.7-99.7%), respectively. Inter-assay agreement was almost perfect (κ = 0.96). Concordance was perfect in samples with high viral loads (cycle threshold < 30). The average time to a positive result on RT-LAMP was 17 min. HG COVID-19 RT-LAMP is a reliable molecular diagnostic kit for detecting SARS-CoV-2, and its performance is comparable to that of RT-PCR. Shorter turnaround times and the possibility of performing molecular diagnostics in the point-of-care setting make it a valuable option for facilities without sophisticated laboratory equipment.

Outbreak of SARS-CoV-2 Lineage 20I/501Y.V1 in a Nursing Home Underlines the Crucial Role of Vaccination in Both Residents and Staff.

Elderly residents in nursing homes are at very high risk of life-threatening COVID-19-related outcomes. In this report, an epidemiological and serological investigation of a SARS-CoV-2 outbreak in an Italian nursing home is described. Among the residents, all but one (19/20) were regularly vaccinated against SARS-CoV-2. In mid-February 2021, a non-vaccinated staff member of the nursing home was diagnosed with the SARS-CoV-2 infection. Following the outbreak investigation, a total of 70% (14/20) of residents aged 77-100 years were found positive. The phylogenetic analysis showed that the outbreak was caused by the SARS-CoV-2 variant of concern 202012/01 (the so-called "UK variant"). However, all but one positive subjects (13/14) were fully asymptomatic. The only symptomatic patient was a vaccinated 86-year-old female with a highly compromised health background and deceased approximately two weeks later. The subsequent serological investigation showed that the deceased patient was the only vaccinated subject that did not develop the anti-spike protein antibody response, therefore being likely a vaccine non-responder. Although the available mRNA SARS-CoV-2 vaccine was not able to prevent several asymptomatic infections, it was able to avert most symptomatic disease cases caused by the SARS-CoV-2 variant of concern 202012/01 in nursing home residents.

Comparative diagnostic performance of rapid antigen detection tests for COVID-19 in a hospital setting.

BACKGROUND: The availability of accurate and rapid diagnostic tools for COVID-19 is essential for tackling the ongoing pandemic. Our study aimed to quantify the performance of available antigen-detecting rapid diagnostic tests (Ag-RDTs) in a real-world hospital setting. METHODS: In this retrospective analysis, the diagnostic performance of 7 Ag-RDTs was compared with real-time reverse transcription quantitative polymerase chain reaction assay in terms of sensitivity, specificity and expected predictive values. RESULTS: A total of 321 matched Ag-RDTreal-time reverse transcription quantitative polymerase chain reaction samples were analyzed retrospectively. The overall sensitivity and specificity of the Ag-RDTs was 78.7% and 100%, respectively. However, a wide range of sensitivity estimates by brand (66.0%-93.8%) and cycle threshold (Ct) cut-off values (Ct <25: 96.2%; Ct 30-35: 31.1%) was observed. The optimal Ct cut-off value that maximized sensitivity was 29. CONCLUSIONS: The routine use of Ag-RDTs may be convenient in moderate-to-high intensity settings when high volumes of specimens are tested every day. However, the diagnostic performance of the commercially available tests may differ substantially.

No evidence of SARS-CoV-2 in hospitalized patients with severe acute respiratory syndrome in five Italian hospitals from 1st November 2019 to 29th February 2020.

BACKGROUND: On 9th January 2020, China CDC reported a novel coronavirus (later named SARS-CoV-2) as the causative agent of the coronavirus disease 2019 (COVID-19). Identifying the first appearance of virus is of epidemiological importance to tracking and mapping the spread of SARS-CoV-2 in a country. We therefore conducted a retrospective observational study to detect SARS-CoV-2 in oropharyngeal samples collected from hospitalized patients with a Severe Acute Respiratory Infection (SARI) enrolled in the DRIVE (Development of Robust and Innovative Vaccine Effectiveness) study in five Italian hospitals (CIRI-IT BIVE hospitals network) (1st November 2019 - 29th February 2020). OBJECTIVES: To acquire new information on the real trend in SARS-CoV-2 infection during pandemic phase I and to determine the possible early appearance of the virus in Italy. MATERIALS AND METHODS: Samples were tested for influenza [RT-PCR assay (A/H1N1, A/H3N2, B/Yam, B/Vic)] in accordance with the DRIVE study protocol. Subsequently, swabs underwent molecular testing for SARS-COV-2. [one-step real-time multiplex retro-transcription (RT) PCR]. RESULTS: In the 1683 samples collected, no evidence of SARS-CoV-2 was found. Moreover, 28.3% (477/1683) of swabs were positive for influenza viruses, the majority being type A (358 vs 119 type B). A/H3N2 was predominant among influenza A viruses (55%); among influenza B viruses, B/Victoria was prevalent. The highest influenza incidence rate was reported in patients aged 0-17 years (40.3%) followed by those aged 18-64 years (24.4%) and ≥65 years (14.8%). CONCLUSIONS: In Italy, some studies have shown the early circulation of SARS-CoV-2 in northern regions, those most severely affected during phase I of the pandemic. In central and southern regions, by contrast no early circulation of the virus was registered. These results are in line with ours. These findings highlight the need to continue to carry out retrospective studies, in order to understand the epidemiology of the novel coronavirus, to better identify the clinical characteristics of COVID-19 in comparison with other acute respiratory illnesses (ARI), and to evaluate the real burden of COVID-19 on the healthcare system.

How relevant is the HIV low level viremia and how is its management changing in the era of modern ART? A large cohort analysis.

BACKGROUND: It is still unclear what might be the best management of people living with HIV (PLWHIV) with low level viremia (LLV) despite being on antiretroviral treatment (ART). OBJECTIVES: Aim of our study is to describe the clinical management of PLWHIV with LLV followed in a large cohort. STUDY DESIGN: Retrospective cohort study. RESULTS: We included 1607 adult patients over a three-year period (2015-2017). Follow up continued until June, 30th 2019 or last available visit. We observed a low incidence of LLV (0.9 % in 2015, 0.7 % in 2016 and 0.4 % in 2017), with a total of 21 patients with persistent LLV (pLLV), i.e. two consecutive HIV-RNA determinations of 50-500 copies/ml after at least 4 months of viral suppression. Among them, 12 had low compliance to treatment. Genotype resistance test (GRT) was performed in 14 patients and demonstrated at least one resistance mutation in 85.7 %. We described three categories of patients with pLLV: i) those whose ART regimen was not adequate based on GRT; ii) those with presumed suboptimal drug exposure, consequence of low adherence and/or drug-drug interactions and iii) those in which pLLV remained unexplained. For the first two categories, optimization or intensification of ART regimen led to viral suppression in >80 % of patients. We observed only 2 (9.5 %) virological failures and 1 (4.8 %) persistence of LLV in patients who did not switch ART. CONCLUSIONS: In our cohort, the rate of LLV showed a decline in most recent years. Adherence and previous GRT should be carefully considered with the aim of further reducing the phenomenon.

Analysis of a 3-months measles outbreak in western Liguria, Italy: Are hospital safe and healthcare workers reliable?

BACKGROUND: From January 2017 to June 2018 more than 7000 measles cases were reported in Italy, of which more than 400 among unvaccinated healthcare workers. We described a measles outbreak occurred in Western Liguria, Italy, characterized by a high involvement of healthcare workers and hospital visitors. METHODS: Suspected measles cases and data regarding vaccination status and clinical management of the patients were collected by reviewing 3 different surveillance systems: the routine mandatory notification system, the National Integrated Surveillance System for Measles and Rubella and the regional reference laboratory for measles diagnosis. RESULTS: Thirty-six cases were reported, with a median age of 31 years and >95% in unvaccinated subjects. One death occurred, 15 cases were hospitalized. Hospital transmission was confirmed or suspected in 12 cases; amongst this cases, 5 were healthcare workers (a gynaecologist, an obstetric nurse, a radiologist, a physiotherapist and a nurse working in an infectious disease ward), all certified unvaccinated. Phylogenetic analysis revealed the circulation of a single B3 genotype variant. CONCLUSIONS: Our experience highlighted the key role of nosocomial transmission and the need for targeted strategies, in particular (i) to implement a measles catch-up immunization campaign in susceptible groups, especially in healthcare workers, (ii) to intensify the check of immunisation status of healthcare workers and to offer vaccination for those who need it, (iii) to improve timeliness and completeness of surveillance systems. Efforts are needed to guarantee the safety of the hospital and the reliability of the healthcare workers. Only high vaccination coverage among HCWs can prevent the diffusion of measles in the hospital setting.

Overview of hepatitis B virus mutations and their implications in the management of infection.

Hepatitis B virus (HBV) affects approximately two billion people worldwide and more than 240 million people in the world are currently chronic carrier that could develop serious complications in the future, like liver cirrhosis and hepatocellular carcinoma. Although an extended HBV immunization program is being carried out since the early '80s, representing effective preventive measure, leading to a dramatic reduction of HBV hepatitis incidence, globally HBV infection still represents a major public health problem. The HBV virus is a DNA virus belongs to the Hepadnaviridae family. The HBV-DNA is a circular, partial double strand genome. All coding information is on the minus DNA strand and it is organized into four open reading frames. Despite hepatitis B virus is a DNA virus, it has a high mutation rate due to its replicative strategy, that leads to the production of many non-identical variants at each cycle of replication. In fact, it contains a polymerase without the proofreading activity, and uses an RNA intermediate (pgRNA) during its replication, so error frequencies are comparable to those seen in retroviruses and other RNA viruses rather than in more stable DNA viruses. Due to the low fidelity of the polymerase, the high replication rate and the overlapping reading frames, mutations occur throughout the genome and they have been identified both in the structural and not structural gene. The arise of mutations being to develop of a whole of viral variants called "quasi-species" and the prevalent population, which favors virus replication, was selected by viral fitness, host's immune pressure and external pressure, i.e., vaccination or antiviral therapy. Naturally occurring mutations were found both in acute and chronic subjects. In the present review we examine and discuss the most recent available data about HBV genetic variability and its significance.

Seroprevalence and vaccination coverage of vaccine-preventable diseases in perinatally HIV-1-infected patients.

BACKGROUND: Even in the era of highly active antiretroviral therapy (HAART), HIV-infected subjects are at higher risk of complications from vaccine-preventable diseases than those uninfected. The current international guidelines strongly recommend that these patients should receive all the routine childhood vaccinations. Although these children represent an appropriate target for immunization, the available data indicate suboptimal coverage rates. METHODS: To evaluate seroprotection/seropositivity rates and vaccination coverage against the common vaccine-preventable diseases, all patients with vertically transmitted HIV-1 infection who attended San Martino Hospital were enrolled. Blood samples were collected for testing antibodies against diphtheria, tetanus, hepatitis A and B viruses by Enzyme-Linked ImmunoSorbent Assay and polioviruses by microneutralization test. In order to assess immunization coverage, retrospectively was recorded the vaccination history collecting data from Regional Immunization Database. RESULTS: A total of 39 perinatally HIV-1 infected patients were included in the study. At the time of serum was obtained, the mean age was 18,1 years (range: 6-28). The median CD4+ T-lymphocyte count was 702 cells/mm(3) (2-1476 cells/mm(3)). Twenty-nine (74.4%) patients were found with HIV RNA load < 50 copies/mL. The proportion of subjects with protective anti-tetanus and anti-HBs were 43.6% and 30.8%, respectively. Seroprotection rates about 20% against rubella and measles were found, less than 20% against all the other antigens investigated. In particular, all patients resulted susceptible to mumps. High immunization rates were observed for polio and HBV (100% and 92.3%, respectively) and suboptimal for diphtheria-tetanus (84.6%). For the other recommended vaccines the rates were generally low. None of the patients received varicella vaccine doses. CONCLUSIONS: As in the HAART era the vertically acquired HIV infection has become a chronic treatable disease, the vaccine-induced long-term protection plays an increasingly significant role; despite good initial response to primary vaccination, subsequent decline and loss of detectable antibodies may be prevented by additional strategies for booster doses of vaccines in adolescents and young adults.

Prevalence of HIV-1 Subtypes and Drug Resistance-Associated Mutations in HIV-1-Positive Treatment-Naive Pregnant Women in Pointe Noire, Republic of the Congo (Kento-Mwana Project).

The Kento-Mwana project was carried out in Pointe Noire, Republic of the Congo, to prevent mother-to-child HIV-1 transmission. To determine the prevalence of different subtypes and transmitted drug resistance-associated mutations, 95 plasma samples were collected at baseline from HIV-1-positive naive pregnant women enrolled in the project during the years 2005-2008. Full protease and partial reverse transcriptase sequencing was performed and 68/95 (71.6%) samples were successfully sequenced. Major mutations to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors were detected in 4/68 (5.9%), 3/68 (4.4%), and 2/68 (2.9%) samples, respectively. Phylogenetic analysis of HIV-1 isolates showed a high prevalence of unique recombinant forms (24/68, 35%), followed by CRF45_cpx (7/68, 10.3%) and subsubtype A3 and subtype G (6/68 each, 8.8%). Although the prevalence of transmitted drug resistance mutations appears to be currently limited, baseline HIV-1 genotyping is highly advisable in conjunction with antiretroviral therapy scale-up in resource-limited settings to optimize treatment and prevent perinatal transmission.

Discordances with HIV-1 RNA quantitative determinations by three commercial assays in Pointe Noire, Republic of Congo.

Accurate HIV-1 RNA quantitation is required to support the scale up of antiretroviral therapy in African countries. Extreme HIV-1 genetic variability in Africa may affect the ability of commercially available assays to detect and quantify HIV-1 RNA accurately. The aim of this study was to compare three real-time PCR assays for quantitation of plasma HIV-1 RNA levels in patients from the Republic of Congo, an area with highly diversified HIV-1 subtypes and recombinants. The Abbott RealTime HIV-1, BioMérieux HIV-1 EasyQ test 1.2 and Cobas AmpliPrep/Cobas TaqMan HIV-1 1.0 were compared for quantitation of HIV-1 RNA in 37 HIV-1 seropositive pregnant women enrolled in the Kento-Mwana project for prevention of mother-to-child transmission in Pointe-Noire, Republic of Congo. The sample panel included a variety of HIV-1 subtypes with as many as 21 (56.8%) putative unique recombinant forms. Qualitative detection of HIV-1 RNA was concordant by all three assays in 33/37 (89.2%) samples. Of the remaining 4 (10.8%) samples, all were positive by Roche, three by Abbott and none by BioMérieux. Differences exceeding 1Log in positive samples were found in 4/31 (12.9%), 10/31 (32.3%) and 5/31 (16.1%) cases between Abbott and BioMérieux, Roche and BioMérieux, and Abbott and Roche, respectively. In this sample panel representative of highly polymorphic HIV-1 in Congo, the agreement among the three assays was moderate in terms of HIV-1 RNA detectability and rather inconsistent in terms of quantitation.

Epidemiology of HBV S-gene mutants in the Liguria Region, Italy: Implications for surveillance and detection of new escape variants.

HBV surface antigen (HBsAg) variants may impair diagnosis or allow the virus to escape vaccine-induced immunity and their circulation in the population can represent a Public Health threat. Their prevalence, however, is not yet completely established. Evidence indicates that amino acid substitutions within HBsAg can lead to conformational changes which allow mutated HBV to escape the vaccine-induced antibodies used in the screening tests. In such scenario, the aim of this study was to investigate the prevalence of HBV S-Gene escape mutants by sequencing the gene in a cohort of Ligurian patients monitored for viral load, genotype and drug resistance and to evaluate the risk of false negative HBsAg detection by routine screening tests. From 2007 to 2011, in 256 consecutive samples from Ligurian HBV positive patients sequencing assay for detection of RT/S-Gene mutations using Trugene HBV Genotyping kit (Siemens Healthcare Diagnostics Inc., Tarrytown, NY) was performed. Serological HBV tests and viral load were also performed. Analyzed sequences revealed G145R mutation in 8/256 (3.1%) examined sequences, it was alone in 5 patients and accompanied by other HBsAg mutations in 3 samples. HBsAg resulted undetectable by 3 of the 8 samples, derived from patients with multiple mutations: T126I-T131A-C139Y-E/D144G, T126I-M133L, and P120Q-T126I. The emergence of these mutants, at least the G145R, has already been addressed as a public health concern because of its capability of escaping the immune system. In the present study we point out a second aspect connected with their existence and with similar potential negative impact on public health, that is their capability of escape punctual detection.