Multiplex Nucleic Acid Amplification Tests Research Articles

State of the Art for Diagnosis of Bacterial Vaginosis.

Bacterial vaginosis (BV) is the most common cause of vaginal discharge among reproductive-age women. It is associated with multiple adverse health outcomes, including increased risk of acquisition of HIV and other sexually transmitted infections (STIs), in addition to adverse birth outcomes. While it is known that BV is a vaginal dysbiosis characterized by a shift in the vaginal microbiota from protective Lactobacillus species to an increase in facultative and strict anaerobic bacteria, its exact etiology remains unknown. The purpose of this minireview is to provide an updated overview of the range of tests currently used for the diagnosis of BV in both clinical and research settings. This article is divided into two primary sections: traditional BV diagnostics and molecular diagnostics. Molecular diagnostic assays, particularly 16S rRNA gene sequencing, shotgun metagenomic sequencing, and fluorescence in situ hybridization (FISH), are specifically highlighted, in addition to multiplex nucleic acid amplification tests (NAATs), given their increasing use in clinical practice (NAATs) and research studies (16S rRNA gene sequencing, shotgun metagenomic sequencing, and FISH) regarding the vaginal microbiota and BV pathogenesis. We also provide a discussion of the strengths and weaknesses of current BV diagnostic tests and discuss future challenges in this field of research.

HIV prevalence and associated risk factors among young tertiary student men who have sex with men (MSM) in Nairobi, Kenya: a respondent-driven sampling survey

BackgroundYoung men who have sex with men (MSM), are a key population at higher risk of HIV infection yet they are underrepresented in research. We conducted a bio-behavioral survey to estimate HIV prevalence and associated risk factors among tertiary student MSM (TSMSM) in Nairobi, Kenya.MethodsBetween February and March 2021, 248 TSMSM aged ≥ 18 years who reported sex with another man in the past year participated in a respondent-driven sampling (RDS) based cross-sectional survey. Participants completed an electronically self-administered behavioral survey and provided a blood sample for HIV antibody testing, alongside urine, anorectal and oropharyngeal swabs for pooled testing of sexually transmitted infections using a multiplex nucleic acid amplification test. RDS-Analyst v.0.72 and Stata v.15 software were used for data analysis. Differences in proportions were examined using chi-square (χ2) test, and unweighted multivariate logistic regression was used to assess factors associated with HIV infection.ResultsHIV prevalence among study participants was 8.3%, whereas the weighted prevalence was 3.6% (95% CI: 1.3–6.0%). Median ages of participants, and at self-reported first anal sex with a man were 21(interquartile range [IQR] 20-22) and 18 (IQR 17-19) years, respectively. A majority (89.3%) of TSMSM owned a smart phone, 46.5% had ever used a geosocial networking app for MSM such as Grindr ® to find a sex partner, and a third (33.6%) met their last sex partner online. Almost three-quarters (71.3%) had > 1 male sex partner in the year before the survey. A third (34.3%) did not use condoms with their last sex partner, 21.2% received money from their last sex partner and 40.9% had taken alcohol/another drug during their last sexual encounter. HIV infection was associated with studying in private institutions (adjusted odds ratio[AOR] = 6.0; 95% confidence intervals [CI] : 1.2–30.0, p = 0.027), preferring a sex partner of any age—younger, same or older (AOR = 5.2; 95 CI: 1.1–25.2, p = 0.041), last sex partner being > 25 years (AOR = 6.4; 95% CI: 1.2–34.6, p = 0.030), meeting the last sex partner online (AOR = 4.2; 95% CI; 1.1–17.0, p = 0.043) and testing positive for Neisseria gonorrhea (AOR = 7.8; 95% CI: 2.0–29.9, p = 0.003).ConclusionsHIV prevalence among TSMSM in Nairobi is alarmingly high, demonstrating a need for tailored prevention and control interventions for this young key population.

Prevalence of five curable sexually transmitted infections and associated risk factors among tertiary student men who have sex with men in Nairobi, Kenya: a respondent-driven sampling survey†.

Young men who have sex with men (MSM) are a key population at high risk of sexually transmitted infections (STIs). We conducted a respondent-driven sampling (RDS) bio-behavioural survey to estimate the prevalence of five curable STIs: chlamydia, gonorrhoea, syphilis, trichomoniasis and Mycoplasma genitalium infection, and associated risk factors among tertiary student MSM (TSMSM) in Nairobi, Kenya. Between February and March 2021, we recruited 248 TSMSM aged ≥18years who self-reported engaging in anal and/or oral sex with another man in the past year. Samples collected included urine, anorectal and oropharyngeal swabs for pooled Chlamydia trachomatis , Mycoplasma genitalium , Neisseria gonorrhoeae and Trichomonas vaginalis testing using multiplex nucleic acid amplification tests, and venous blood for serological Treponema pallidum screening and confirmation of current infection. Participants self-completed a behavioural survey on a REDCap digital platform. Data analysis was done using RDS-Analyst (v0.72) and Stata (v15). Differences in proportions were examined using the chi-squared (χ 2 ) test, and unweighted multivariate logistic regression was used to assess factors associated with STI prevalence. RDS-adjusted prevalence rates of at least one of the five STIs, chlamydia, gonorrhoea, Mycoplasma genitalium infection, trichomoniasis and latent syphilis were 58.8%, 51.0%, 11.3%, 6.0%, 1.5% and 0.7%, respectively. Factors independently associated with STI prevalence were inconsistent condom use (adjusted odds ratio (AOR)=1.89, 95% confidence interval (CI): 1.03-3.47, P =0.038) and the last sex partner being a regular partner (AOR=2.35, 95% CI: 1.12-4.92, P =0.023). STI prevalence among TSMSM in Nairobi, Kenya, is disturbingly high, demonstrating urgent need for tailored testing, treatment and prevention interventions for this population.

Comparative analysis of three multiplex platforms for the detection of respiratory viral pathogens.

Acute viral respiratory infections are a major health burden in children worldwide. In recent years, rapid and sensitive multiplex nucleic acid amplification tests (NAATs) have replaced conventional methods for routine virus detection in the clinical laboratory. We compared BioFire® FilmArray® Respiratory Panel (FilmArray V1.7), Luminex NxTag® Respiratory Pathogen Panel (NxTag RPP) and Applied Biosystems TaqMan Array Card (TAC) for the detection of eight viruses in pediatric respiratory specimens. Results from the three platforms were analyzed with a single-plex real-time RT-PCR (rRT-PCR) assay for each virus. Of the 170/210 single-plex virus-positive samples, FilmArray detected a virus in 166 (97.6%), TAC in 163 (95.8%) and NxTag RPP in 160 (94.1%) samples. The Positive Percent Agreement (PPA) of FilmArray, NxTag RPP and TAC was highest for influenza B (100%, 100% and 95.2% respectively) and lowest for seasonal coronaviruses on both FilmArray (90.2%) and NxTag RPP (81.8%), and for parainfluenza viruses 1- 4 on TAC (84%). The Negative Percent Agreement (NPA) was lowest for rhinovirus/enterovirus (92.9%, 96.7% and 97.3%) on FilmArray, NxTag RPP and TAC respectively. NPA for all three platforms was highest (100%) for both parainfluenza viruses 1- 4 and influenza A and B, and 100% for human metapneumovirus with TAC as well. All three multiplex platforms displayed high overall agreement (>90%) and high NPA (>90%), while PPA was pathogen dependent and varied among platforms; high PPA (>90%) was observed for FilmArray for all eight viruses, TAC for six viruses and NxTag RPP for 4 viruses.

Clinical evaluation of the BioFire Global Fever Panel for the identification of malaria, leptospirosis, chikungunya, and dengue from whole blood: a prospective, multicentre, cross-sectional diagnostic accuracy study

Acute febrile illness is a common presentation for patients at hospitals globally. Assays that can diagnose a variety of common pathogens in blood could help to establish a diagnosis for targeted disease management. We aimed to evaluate the performance of the BioFire Global Fever Panel (GF Panel), a multiplex nucleic acid amplification test performed on whole blood specimens run on the BioFire FilmArray System, in the diagnosis of several pathogens that cause acute febrile illness. We did a prospective, multicentre, cross-sectional diagnostic accuracy study to evaluate the GF Panel. Consenting adults and children older than 6 months presenting with fever in the previous 2 days were enrolled consecutively in sub-Saharan Africa (Ghana, Kenya, Tanzania, Uganda), southeast Asia (Cambodia, Thailand), central and South America (Honduras, Peru), and the USA (Washington, DC; St Louis, MO). We assessed the performance of six analytes (chikungunya virus, dengue virus [serotypes 1-4], Leptospira spp, Plasmodium spp, Plasmodium falciparum, and Plasmodium vivax or Plasmodium ovale) on the GF Panel. The performance of the GF Panel was assessed using comparator PCR assays with different primers followed by bidirectional sequencing on nucleic acid extracts from the same specimen. We calculated the positive percent agreement and negative percent agreement of the GF Panel with respect to the comparator assays. This study is registered with ClinicalTrials.gov, NCT02968355. From March 26, 2018, to Sept 30, 2019, 1965 participants were enrolled at ten sites worldwide. Of the 1875 participants with analysable results, 980 (52·3%) were female and the median age was 22 years (range 0-100). At least one analyte was detected in 657 (35·0%) of 1875 specimens. The GF Panel had a positive percent agreement for the six analytes evaluated as follows: chikungunya virus 100% (95% CI 86·3-100), dengue virus 94·0% (90·6-96·5), Leptospira spp 93·8% (69·8-99·8), Plasmodium spp 98·3% (96·3-99·4), P falciparum 92·7% (88·8-95·6), and P vivax or P ovale 92·7% (86·7-96·6). The GF Panel had a negative percent agreement equal to or greater than 99·2% (98·6-99·6) for all analytes. This 1 h sample-to-answer, molecular device can detect common causative agents of acute febrile illness with excellent positive percent agreement and negative percent agreement directly in whole blood. The targets of the assay are prevalent in tropical and subtropical regions globally, and the assay could help to provide both public health surveillance and individual diagnoses. BioFire Defense, Joint Project Manager for Medical Countermeasure Systems and US Army Medical Materiel Development Activity, and National Institute of Allergy and Infectious Diseases.

The Using of Multiplex RT-qPCR for Pooled Samples to Detect Hepatitis B Virus, Hepatitis C Virus, Human Immunodeficiency Virus within Iraqi Blood Donors

Background: The blood of blood donors is screening for hepatitis B virus (HBV), hepatitis C virus (HCV), and human immunodeficiency virus (HIV) infection by enzyme immunoassay (EIA) in the National Center of Blood Bank in Baghdad. The residual risk of EIA negative samples is not estimated till now in Iraq. Objective: To detect HBV, HCV and HIV viruses within seronegative plasma of blood donors by a commercially available multiplex nucleic acid amplification tests (NAT) with mini-pooling system. Methods: One thousand (1000) blood donors were screened by EIA revealed negative results then NAT was performed on pools of samples, each pool contain ten seronegative plasmas (MP10) i.e., 100 minipools. Results: The detected positive minipools 10 seronegative plasmas by NAT for HBV, HCV and HIV were 3 MP10. Conclusion: The use of NAT appeared to be sensitive and reliable to detect occult HBV and overcome the seroconversion problem related with HCV and HIV within seronegative plasmas of blood donors. Therefore, the implementation of NAT with mini-pooling in addition to serological tests for routine blood donor screening will improve and ensure the safety of blood transfusion in Iraq. Keywords: Blood transfusion, widow period, multiplexes PCR Citation: Majid GS, Abdulamir AS, Ahmed AM, Aufi IM, Abdullah OI. The using of multiplex RT-qPCR for pooled samples to detect hepatitis B virus, hepatitis C virus, human immunodeficiency virus within Iraqi blood donors. Iraqi JMS. 2021; 19(1): 99-106. doi: 10.22578/IJMS.19.1.13

Leukocyturia and Sexually Transmitted Infections: A Cohort Study of 2236 Asymptomatic Adults

Background: Sexually transmitted infections (STIs) can cause leukocyturia in asymptomatic patient. We aimed to evaluate leukocyturia prevalence in asymptomatic aircrew and STIs proportion in leukocyturia. Methods: The LEUCO survey was a prospective, monocentre cohort study among aircrew, done at aeromedical center in France. Aircrews were included during five months. Systematic dipstick urinalysis leukocyturia result was collected. Participants with leukocyturia were proposed to perform a Chlamydia trachomatis, Neisseria gonorrhoeae, Mycoplasma genitalium and Trichomonas vaginalis, nucleic acid amplification test (NAAT). We calculated leukocyturia prevalence and STIs proportion among this. We used a logistic regression model to search for associations between leukocyturia and variables collected. This study was registered in our Institutional Review Board, number IRB 0011873202022. Findings: Between Oct 14, 2019, and Mar 13, 2020, 2236 participants performed dipstick urinalysis, of whom 127 (5.7%) had a leukocyturia. The prevalence of leukocyturia was significantly different between men, 1.9% (1.3−2.6) and women, 28.1% (23.3−33.3) (p<0.001). Female gender was associated with leukocyturia OR 21.6 (13.1−36.4) (p<0.001). In men with leukocyturia, NAAT positivity rate was very high in the 18-24 age group, 28.6% (3.7−71.0) and in the 25-34 age group, 20.0% (0.5-71.6). In women with leukocyturia, it was high in the 18-24 age group, 16.7% (4.7−37.4) and in the 25-34 age group, 18.2% (2.3−51.8). Interpretation Leukocyturia is rare in asymptomatic subjects. Patients with leukocyturia under 35 years old have high proportion of STIs. Multiplex NAAT has utility for STIs diagnosis in asymptomatic subjects under 35 years old presenting a leukocyturia. Trial Registration: Trial number IRB 0011873202022. Funding: French Army Health Service Declaration of Interest: None to declare. Ethical Approval: The survey received a favorable ethical opinion from the clinical trials validation commission of the Sainte-Anne military hospital Toulon, France, registered as an Institutional Review Board (IRB 0011873202022 ).

Application of Multiplex Molecular Assays in the Diagnosis of Acute Respiratory Infectious Diseases

Acute respiratory tract infections (ARTIs) are the most common diseases worldwide and an important cause of morbidity and mortality. Upper and lower respiratory tract infections are caused by a variety of bacterial, viral, and fungal pathogens. Clinical diagnosis of respiratory tract infections is challenging because of indistinguishable symptoms. Laboratory diagnosis is performed by serology, culture, electron microscopy, and immunological antigen detection assays. These conventional diagnostic procedures are time consuming, lack sensitivity, require special laboratory setup, and well-trained staff. In the clinical laboratories, multiplex molecular nucleic acid amplification tests (NAATs) are continuously replacing conventional diagnostic methods. This review summarizes and discusses the availability, clinical use, advantages and disadvantages of multiplex molecular methods in the detection and identification of ARTI pathogens. The multiplex molecular assays can simultaneously detect 20 or more bacterial and viral pathogens and have the advantage of increased sensitivity, specificity, and rapid turn-around time. These assays are helpful in syndromic based testing in high risk patient population, particularly those who are immunocompromised, hospitalized and/or seen in the emergency department. Limitations of multiplex molecular assays include inability to detect all the possible pathogens that can be present, not being able to differentiate between asymptomatic carriers and true infections, and sometimes results are difficult to interpret. Furthermore, additional testing may be required as these assays do not provide any information regarding antimicrobial susceptibility profile. Rapid and accurate diagnosis of respiratory tract infections leads to better treatment decisions, reduction in the further diagnostic procedures, length of hospital stay, better infection control measures, and associated healthcare costs.

Optimal Timing of Repeat Multiplex Molecular Testing for Respiratory Viruses.

Determining whether and when multiplex nucleic acid amplification tests (NAATs) for respiratory viruses should be repeated is difficult. We analyzed 5 years of results for a multiplex NAAT targeting 14 respiratory viruses, to determine how often repeat tests were ordered and the time period in which results were likely to change. Results for NAATs performed on nasopharyngeal specimens and repeated within 90 days after initial testing were analyzed. Logistic regression models were used to compare time periods between tests with respect to the odds of a change in the sample result. During the study period, 21,819 nasopharyngeal specimens from 16,779 individuals were submitted. Of these, 8,807 samples (40%) were positive for at least one viral pathogen. Among this cohort, 2,583 specimens (12%) collected from 1,473 patients (9%) were repeat tests performed within 90 days after an initial test. If repeated within 90 days, 71% of tests (1,833 tests) did not have a change in result. Initially negative tests typically remained negative, whereas initially positive tests mostly remained positive until 11 to 15 days. The odds of result change plateaued after 20 days. The odds of result change for tests repeated within 20 days were only 0.52 times the odds (95% confidence interval, 0.43 to 0.62) for those repeated at 21 to 90 days (P < 0.001). Multiplex tests for respiratory viruses that are repeated within short periods lead to redundant results at additional costs. Repeat testing of nasopharyngeal specimens before 20 days demonstrates little change. These results provide a vital component for use in laboratory stewardship to curtail unnecessary respiratory viral testing.

Evaluation of a multiplex PCR assay for detection of respiratory viruses and Mycoplasma pneumoniae in oropharyngeal swab samples from outpatients.

BackgroundRespiratory viruses, such as influenza viruses, initially infect the upper airways but can manifest as severe lower respiratory tract infections in high‐risk patients with significant morbidity and mortality. For syndromic diagnosis, several multiplex nucleic acid amplification tests have been developed for clinics, of which SureX 13 Respiratory Pathogen Multiplex Kit (ResP) can simultaneously detect 13 pathogens directly from airway secretion specimens. The organisms identified are influenza virus A, influenza virus A pdmH1N1 (2009), influenza virus A H3N2, influenza virus B, adenovirus, boca virus, rhinovirus, parainfluenza virus, coronavirus, respiratory syncytial virus, human metapneumovirus, Mycoplasma pneumoniae, and Chlamydia.MethodsThis study provides performance evaluation data of this assay by comparing with pathogen‐specific PCRs from oropharyngeal swab samples.ResultsTen pathogens were detected in this assay, of which rhinovirus, adenovirus, and influenza virus A pdmH1N1 (2009) were the most common. The overall agreement between the ResP and the comparator tests was 93.8%. The ResP demonstrated 86.5% agreement for positive results and 97.8% agreement for negative results.ConclusionThe ResP assay demonstrated a highly concordant performance comparing with pathogen‐specific PCRs for detection of respiratory pathogens in oropharyngeal swabs from outpatients and could aid in the diagnosis of respiratory infections in a variety of clinical scenarios.

Viral respiratory infections diagnosed by multiplex polymerase chain reaction in pediatric patients.

Syndromic diagnosis by multiplex nucleic acid amplification tests is the most practical approach to respiratory tract infections since the symptoms are rarely agent‐specific. The aim of this study was to investigate the respiratory viruses in children admitted to a university hospital with acute respiratory tract infection during the last 8 years by a multiplex polymerase chain reaction (PCR) assay. A total of 3162 respiratory samples collected from children between April 2011 and April 2018 tested by a multiplex real‐time PCR assay. Two different commercial assays were used during the study period, "AusDiagnostics/Respiratory Pathogens 12 (AusDiagnostics)" used between April 2011 and December 2015, which changed to "Fast Track Diagnostics/Respiratory Pathogens 21 (Fast Track Diagnostics)" after January 2016 to cover more viruses. Nucleic acid extraction was done by EZ1 Advanced XL platform (QIAGEN). Respiratory pathogens detected in 1857 of the 3162 (58.7%) samples. The most prevalent viruses during the 8‐year period were rhinovirus/enterovirus (RV/EV; 36.2%), respiratory syncytial virus (RSV; 19%), and influenza virus A/B (14.7%). Rhinovirus was the main contributor to the RV/EV group as shown by the assay used during the 2016‐2018 period. RV/EV and adenoviruses detected throughout the year. Influenza virus was most frequently detected during January to March when both RSV and metapneumovirus were also in circulation. The coinfection percentage was 10.2%. Rhinovirus was the most common virus in coinfections while RSV plus rhinovirus/enterovirus were the most frequent combination. RSV and metapneumovirus showed a similar seasonal distribution to the influenza virus, which made it necessary to use a virological diagnostic assay during the influenza season.

NATSpert ID TripleH: A novel individual donor multiplex nucleic acid amplification test to reduce risk of transfusion-transmitted infections: Two-year experience of a blood bank in central India

Background: In India serological screening is mandatory for all donated blood for hepatitis B virus (HBV), hepatitis C virus (HCV) and human immunodeficiency virus (HIV) while Nucleic Acid Amplification Testing (NAT) is not a mandatory screening test. There is a risk of transfusion-transmitted infections (TTI) during window period using serology. Aims and Objectives: The aim of this study was to analyse NAT screening using an indigenously developed, Indian manufactured Individual Donor NAT (ID-NAT) and compare it with serology. Materials and Methods: All blood donations between June 2017-March 2019 were screened serologically for HBV, HCV and HIV at an Indian blood bank. Blood donations also underwent ID-NAT screening using the NATSpert ID TripleH detection assay based on real time PCR. The results were analysed to identify yield cases. Results: In the study, 30,772 blood donor samples were screened serologically out of which 214 were reactive. 30,558 serologically non-reactive blood donations and 77 randomly selected, serologically reactive blood donations were screened using NATSpert. Out of 30,635, 85 donor samples were reactive on the NATSpert which included 77 serology positive and 8 NAT yield cases. The NAT yield found was 2 each for HBV/HIV and 4 of HCV. Conclusion: The NATSpert ID TripleH offers a statistically significant advantage over EIA in ability to detect TTI in blood donors (P < 0.05, Fishers Exact Test). The NAT yield of 1:3829 was in line with other Indian studies. NATSpert assay will provide a significant improvement in blood safety and offer a cost benefit compared to the imported products.

Rapid multiplex nucleic acid amplification test developed using paper chromatography chip and azobenzene-modified oligonucleotides

Although nucleic acid amplification test (NAT) is widely used for pathogen detection, rapid NAT systems that do not require special and expensive instruments must be developed in order to enable point of care (POC)-NATs, which contribute to early initiation of treatment. As a POC-NAT system, Kaneka DNA chromatography chip (KDCC), developed using DNA tag-bound primer through modified substance, was shown to be suitable for POC testing, due to the rapid detection time, simple procedures, and low manufacturing costs. However, owing to some modifications in primer, the detection performance and amplification speed were shown to be reduced when using KDCC, counteracting the increased speed of detection. To solve these issues and improve the speed of this NAT system, we investigated a better modification substance for KDCC. Here, azobenzene-modified primers were shown to have the highest amplification speed and detection performance in KDCC, of all modifications tested in this study, showing 10-100-fold lower detection limit but maintaining the same reaction time. Additionally, rapid herpes simplex virus detection system with azobenzene modified primers was developed. We believed that this breakthrough will contribute toward enabling greater utilization of POC-NATs for medical care, especially in developing countries and clinics.

Epidemiologic Analysis of Respiratory Viral Infections Mainly in Hospitalized Children and Adults in a Midwest University Medical Center After the Implementation of a 14-Virus Multiplex Nucleic Acid Amplification Test

Objectives: To investigate the etiology of viral respiratory tract infections mainly in hospitalized children and adults over a 12-month consecutive period after implementation of a 14-virus multiplex nucleic acid amplification test. Methods: From January 2014 to January 2015, a total of 2,237 respiratory samples were analyzed with the US Food and Drug Administration–cleared eSensor Respiratory Viral Panel (GenMark Diagnostics, Carlsbad, CA). Results: Of the 2,237 specimens tested, 788 specimens were positive for at least one virus, giving a positivity rate of 35.2%, and because of viral codetection, a total of 862 viral targets were identified. The age groups with the highest positivity rates were the 0- to 1-year (73.5%) and 2- to 6-year (78.4%) age groups. The overall viral codetection rate was 9.1%. Human rhinovirus (HRV) was the most prevalent respiratory virus found in children and adults. The peak of HRV seen in September 2014 represented a combination of HRV and enterovirus D68, 2014 epidemic respiratory infections. Conclusion: The ability to detect a wider range of respiratory viruses gave us a better understanding of the etiology of respiratory infections in our population, particularly for HRV and enhanced our ability to detect viral coinfection.

PWE-064 The Utility of An Enteric Bacterial PCR Panel In Evaluating Patients with Diarrhoea and Raised Faecal Calprotectin

Introduction Faecal calprotectin (FCP) assay has an important role in identifying potential cases of inflammatory bowel disease (IBD). However, a significant proportion of patients presenting with diarrhoea and raised FCP do not prove to have IBD following investigation of whom a proportion have an acute self-limiting gut infection. Enteric pathogens are alternative causes of a raised FCP. Alleviating the need for endoscopy in the patients will save money and reduce patient anxiety. However, conventional laboratory methodologies are time consuming and insensitive in detecting gut infection. Commercial nucleic acid amplification test (NAAT) technology now makes it possible to test stool for a panel of gut pathogens quickly and relatively cheaply. In this pilot study, we evaluated this technology in detecting gut infection amongst patients with a diarrhoeal illness and raised FCP, presenting in the community. Methods Ninety patients with diarrhoea had stool samples submitted from the community to the laboratory for FCP measurement. Samples were anonymised before testing with NAAT (BD MAX™ Enteric Bacterial Panel). The targets of this assay are Salmonella spp. , Campylobacter spp. Shigella ( Shigella spp . and enteroinvasive E. coli ) and verotoxigenic E coli . Samples were stratified by FCP level into sub-groups (n = 30): 150 µg/g stool and analysed for the presence and type of gut infection found. All samples were also cultured using standard media. Results 2/30 samples tested (7%, 95% CI 8–23%) with FCP > 150 µg/g tested positive by NAAT for Campylobacter. No patients with FCP below 150 µg/g had a detectable pathogen. No samples were positive by conventional culture methods. Conclusion This pilot study supports a role for the use of multiplex NAAT in detecting enteric pathogens amongst patients with diarrhoea and raised FCP. Prospective studies are required to evaluate this approach further which offers the potential to refine the IBD referral pathways from primary care and reduce the need for endoscopic and radiological tests. Disclosure of Interest None Declared

Diagnostic performance of near-patient testing for influenza

BackgroundRapid diagnosis of influenza is important for controlling outbreaks and starting antiviral therapy. Direct antigen detection (DAD) is rapid, but lacks sensitivity, whereas nucleic acid amplification testing (NAT) is more sensitive, but also more time-consuming. ObjectivesTo evaluate the performance of a rapid isothermal NAT and two DADs. Study designDuring February–May 2014, we tested 211 consecutive patients with influenza-like illness using a commercial isothermal NAT (Alere™ Influenza A&B) as well as the DAD Sofia® Influenza A+B and BinaxNOW® Influenza A&B for detection of influenza-A and -B virus. RespiFinder-22® a commercial multiplex NAT served as reference test. Serial 10-fold dilutions of influenza-A and -B cell culture supernatants were examined. Another 225 patient samples were tested during December 2014–February 2015. ResultsCompared to RespiFinder-22®, the isothermal NAT Alere™ Influenza A&B, and the DAD Sofia® Influenza A+B and BinaxNOW® Influenza A&B had sensitivities of 77.8%, 59.3% and 29.6%, and specificities of 99.5%, 98.9% and 100%, respectively, for the first 211 patient samples. Alere™ Influenza A&B showed 85.7% sensitivity and 100% specificity in the second cohort. Isothermal NAT was 10-100-fold more sensitive compared to DAD for influenza virus culture supernatants with a lower limit of detection of 5000–50,000 copies/mL. The average turn-around time (TAT) of isothermal NAT and DADs was 15min, but increased to 110min for Alere™ Influenza A&B, 30min for BinaxNOW® Influenza A&B, and 45min for Sofia® Influenza A+B, when analyzing batches of 6 samples. ConclusionSimple sample processing and a TAT of 15min render isothermal NAT Alere™ Influenza A&B suitable for sequential near-patient testing, but the TAT advantage is lost when testing of larger series.

Evaluation of the Luminex xTAG Gastrointestinal Pathogen Panel and the Savyon Diagnostics Gastrointestinal Infection Panel for the detection of enteric pathogens in clinical samples.

Infectious gastrointestinal disease is caused by a diverse array of pathogens, and is a challenging syndrome to correctly diagnose and manage. Conventional laboratory diagnostic methods are often time-consuming and frequently suffer from low detection rates. Two commercial multiplex nucleic acid amplification tests [Luminex xTAG Gastrointestinal Pathogen Panel (GPP) and Savyon Diagnostics Gastrointestinal Infection Panel (GIP)] were applied to 1000 stored diarrhoeal clinical stool samples. The Luminex xTAG GPP and Savyon GIP detected Campylobacter in 42/44 and 44/44 culture-positive samples, Salmonella in 4/4 and 3/4 culture-positive samples, Shigella in 1/1 culture-positive sample, Clostridium difficile toxin in 32/35 ELISA-positive samples, and Giardia in 6/6 wet-preparation-microscopy-positive samples, respectively. When the Luminex GPP assay was used concurrently with conventional methods for 472 clinical samples, it detected Campylobacter in 22/22 culture-positive samples, Salmonella in 1/1 culture-positive sample, Clostridium difficile toxin in 14/14 ELISA-positive samples and Giardia in 4/4 wet-preparation-microscopy-positive samples. The pathogen/toxin detection rate for conventional methods in both sample sets was <10%. The Luminex xTAG GPP detection rate was 24.8% in the stored samples and 32.6% in the concurrently tested samples. The Savyon GIP detection rate was 22.5%. From stored samples, 2.4% of Luminex xTAG GPP detections and 3.1% of Savyon GIP detections could not be confirmed using alternative nucleic acid amplification tests. Enhanced detection rates resulted from increased detection of pathogens routinely sought using conventional methods and were also due to ascertainment of micro-organisms that current testing strategies do not diagnose. Use of multiplex nucleic acid amplification tests will allow clinical laboratories to diagnose infectious gastroenteritis in more patients with diarrhoeal disease by increasing the sensitivity of pathogen detection and by reducing the selective bias of current strategies. The clinical and economic impact of these results warrants further investigation.

Gastrointestinal pathogens detected by multiplex nucleic acid amplification testing in stools of pediatric patients and patients returning from the tropics.

Gastrointestinal infections are caused by a broad spectrum of pathogens. Conventional diagnostic procedures are resource and time consuming due to single pathogen testing, often in different laboratories. We analyzed 312 consecutive stool samples from pediatric patients (n = 127) with gastroenteritis or from adult travelers returning from the tropics with suspected parasite infestation (n = 185) using commercial multiplex nucleic acid amplification testing (NAT) (xTAG gastrointestinal pathogen panel, Luminex) covering 15 diarrhea-causing pathogens. The results of the positive samples and a representative number of negative samples were compared to standard methods, including NAT, direct antigen detection (DAD), bacterial culture and microscopy. Of the 185 samples from adult travelers, 21 (11 %) were multiplexNAT-positive, with enterotoxigenic Escherichia coli (4 %) being the predominant pathogen. Microscopic examination revealed Blastocystis hominis in 23 % not covered by the panel. MultiplexNAT scored positive in 66 pediatric samples (52 %), with rotavirus (27 %) being the most prevalent. All adenovirus-, rotavirus-, Clostridium difficile- and Cryptosporidium-positive samples were confirmed in external laboratories, but only 40 % of norovirus- and 29 % of Giardia-positive samples. Analysis of frozen specimens by bacterial culture showed the highest discrepancies with the multiplexNAT. Our study demonstrates broad detection of relevant gastroenteritis pathogens by multiplexNAT with a short turnaround time. This is important for diagnosis, infection control and empiric management of gastroenteritis patients, but may be selectively complemented by bacterial culture and resistance testing.

A pilot study on screening blood donors with individual-donation nucleic acid testing in China.

Nucleic acid amplification testing (NAT) is not yet obligatory in China for blood donor screening and the risk of enzyme immunoassay (EIA)-negative, NAT-reactive donations in Chinese blood donors has rarely been reported. The aim of this study was to screen a population of Chinese blood donors using a triplex individual-donation (ID)-NAT assay and assess the safety benefits of implementing NAT. Between 1st August, 2010 and 31st December, 2011 all donations at a Chinese blood centre were screened individually using the Procleix® Ultrio® assay, a multiplex NAT assay for the detection of hepatitis B virus (HBV) DNA, hepatitis C virus (HCV) RNA and human immunodeficiency virus-1 (HIV-1) RNA. All donations were also screened for HBsAg, anti-HIV and anti-HCV using two different EIA for each marker. Samples with discordant results between NAT and EIA were further tested with an alternative NAT assay (Cobas® TaqMan®). Potential yield cases (serologically negative/NAT-reactive donors) were further evaluated when possible. During the study period a total of 178,447 donations were screened by NAT and EIA, among which 169 HBV NAT yield cases (0.095%) were detected. No N AT yield cases were found for HIV-1 or HCV. For the HBV NAT yield cases, follow-up results showed that 11 (6.51%) were probable or confirmed HBV window period infections, 5 (2.96%) were chronic HBV carriers and 153 (90.53%) were probable or confirmed occult HBV infections. There was a statistically significant difference between the NAT-positive rates for first-time vs repeat donations (0.472% vs 0.146%, respectively; P<0.001). Our data demonstrate that the potential HBV yield rate was 1:1,056 for blood donations in the Zhejiang province of China. Implementation of NAT will provide a significant increment in safety relative to serological screening alone.

Performance of Different Mono- and Multiplex Nucleic Acid Amplification Tests on a Multipathogen External Quality Assessment Panel

An external quality assessment (EQA) panel consisting of a total of 48 samples in bronchoalveolar lavage (BAL) fluid or transport medium was prepared in collaboration with Quality Control for Molecular Diagnostics (QCMD) (www.qcmd.org). The panel was used to assess the proficiency of the three laboratories that would be responsible for examining the 6,000 samples to be collected in the GRACE Network of Excellence (www.grace-lrti.org). The main objective was to decide on the best-performing testing approach for the detection of influenza viruses A and B, parainfluenza virus types 1 to 3, respiratory syncytial virus (RSV), human metapneumovirus, coronavirus, rhinovirus, adenovirus, Chlamydophila pneumoniae, Mycoplasma pneumoniae, and Legionella pneumophila by nucleic acid amplification techniques (NAATs). Two approaches were chosen: (i) laboratories testing samples using their in-house procedures for extraction and amplification and (ii) laboratories using their in-house amplification procedures on centrally extracted samples. Furthermore, three commercially available multiplex NAAT tests-the ResPlex (Qiagen GmbH, Hilden, Germany), RespiFinder plus (PathoFinder, Maastricht, The Netherlands), and RespiFinder Smart 21 (PathoFinder) tests-were evaluated by examination of the same EQA panel by the manufacturer. No large differences among the 3 laboratories were noticed when the performances of the assays developed in-house in combination with the in-house extraction procedures were compared. Also, the extraction procedure (central versus local) had little effect on performance. However, large differences in amplification efficacy were found between the commercially available tests; acceptable results were obtained by using the PathoFinder assays.