Virological Diagnosis of Central Nervous System Infections by Use of PCR Coupled with Mass Spectrometry Analysis of Cerebrospinal Fluid Samples

Viruses are the leading cause of central nervous system (CNS) infections, ahead of bacteria, parasites, and fungal agents. A rapid and comprehensive virologic diagnostic testing method is needed to improve the therapeutic management of hospitalized pediatric or adult patients. In this study, we assessed the clinical performance of PCR amplification coupled with electrospray ionization-time of flight mass spectrometry analysis (PCR-MS) for the diagnosis of viral CNS infections. Three hundred twenty-seven cerebrospinal fluid (CSF) samples prospectively tested by routine PCR assays between 2004 and 2012 in two university hospital centers (Toulouse and Reims, France) were retrospectively analyzed by PCR-MS analysis using primers targeted to adenovirus, human herpesviruses 1 to 8 (HHV-1 to -8), polyomaviruses BK and JC, parvovirus B19, and enteroviruses (EV). PCR-MS detected single or multiple virus infections in 190 (83%) of the 229 samples that tested positive by routine PCR analysis and in 10 (10.2%) of the 98 samples that tested negative. The PCR-MS results correlated well with herpes simplex virus 1 (HSV-1), varicella-zoster virus (VZV), and EV detection by routine PCR assays (kappa values [95% confidence intervals], 0.80 [0.69 to 0.92], 0.85 [0.71 to 0.98], and 0.84 [0.78 to 0.90], respectively), whereas a weak correlation was observed with Epstein-Barr virus (EBV) (0.34 [0.10 to 0.58]). Twenty-six coinfections and 16 instances of uncommon neurotropic viruses (HHV-7 [n = 13], parvovirus B19 [n = 2], and adenovirus [n = 1]) were identified by the PCR-MS analysis, whereas only 4 coinfections had been prospectively evidenced using routine PCR assays (P < 0.01). In conclusion, our results demonstrated that PCR-MS analysis is a valuable tool to identify common neurotropic viruses in CSF (with, however, limitations that were identified regarding EBV and EV detection) and may be of major interest in better understanding the clinical impact of multiple or neglected viral neurological infections.     

Development and evaluation of a panel of multiplex one-tube nested real time PCR assay for simultaneous detection of 14 respiratory viruses in five reactions

Multiplex real-time quantitative polymerase chain reaction (mRT-qPCR) assay is commonly used to detect respiratory viruses, however, the sensitivity is limited for most reports. A panel of locked nucleic acid based multiplex closed one-tube nested real-time PCR (mOTNRT-PCR) assay consisting of five separate internally controlled RT-qPCR assays was developed for detection of 14 respiratory viruses. The sensitivity and reproducibility of mOTNRT-PCR panel were evaluated using plasmid standards and the specificity was evaluated using clinical samples. The clinical performance of mOTNRT-PCR panel was further evaluated with 468 samples collected from patients with an acute respiratory infection and compared with individual real-time PCR (RT-qPCR) assay. The analytical sensitivities of mOTNRT-PCR panel ranged from 2 to 20 copies/reaction, and no cross-reaction with common respiratory viruses was observed. The coefficients of variation of intra-assay and inter-assay were between 0.35% and 8.29%. Totally 35 clinical samples detected by mOTNRT-PCR assay panel were missed by RT-qPCR and confirmed true positive by sequencing of nested PCR products. The mOTNRT-PCR assay panel provides a more sensitive and high-throughput method for the detection of 14 respiratory viruses.     

Prévalence de l’infection par le parvovirus B19 humain au cours des éruptions fébriles de l’enfant dans le Nord tunisien

The aim of the study is to evaluate the prevalence of specific antibodies anti-human parvovirus B19 (PVB19) immunoglobulin M (IgM) and IgG in children with fever and rash. This study involved 257 children aged from 7 months to 15 years with febrile rash unrelated to measles and rubella (seronegative for IgM). The sera were examined by immunoenzymatic assay. Detection of antibodies of PVB19 was done by enzyme-linked immunosorbent assay (Elisa). In our study, prevalence of immunoglobulin G (IgG) and IgM were 44 and 11.3%, respectively. Clinically, children with positive IgM serology had submitted an erythema infectiosum (13/29 cases), myocarditis (1 case), encephalitis (1 case), severe sickle cell anemia (7 cases), and immunocompromised (7 cases). The incidence rate of viral infection was 11.3%; most of the cases of PVB19 infection occurred between the months of May and August. Incidence was higher in the 10–15 years age group (21%). The prevalence of IgG antibody varied and increased with age, it rises from 38.2% in preschool children (19 months–4 years) to 53.5% in those aged between 4.5 and 15 years, reaching 58% in the 10–15 years age group. The four risk factors of PVB19 infection are: (1) those aged between 4.5 and 9 years, which is the most affected age group (P = 0.0018); (2) female gender in children aged between 19 months and 4 years (P = 0.037); (3) transfusion and (4) immune deficiency (P = 0.022 and P = 0.001, respectively). The study of the prevalence of PVB19 infection shows that viral infection is acquired early in childhood, increases with age; viral transmission is favored by the community life. Because of the widespread vaccination program against measles and rubella, the systematic search of PVB19 in front of eruptive fevers becomes important.     

Epidemiological, molecular, and clinical features of enterovirus respiratory infections in French children between 1999 and 2005

Enteroviruses (EVs) can induce nonspecific respiratory tract infections in children, but their epidemiological, virological, and clinical features remain to be assessed. In the present study, we analyzed 252 EV-related infection cases (median age of subjects, 5.1 years) diagnosed among 11,509 consecutive children visiting emergency departments within a 7-year period in the north of France. EV strains were isolated from nasopharyngeal samples by viral cell culture, identified by seroneutralization assay, and genetically compared by partial amplification and sequencing of the VP1 gene. The respiratory syndromes (79 [31%] of 252 EV infections) appeared as the second most common EV-induced pediatric pathology after meningitis (111 [44%] of 252 cases) (44 versus 31%, P < 10⁻³), contributing to lower respiratory tract infection (LRTI) in 43 (54%) of 79 EV respiratory infection cases. Bronchiolitis was the most common EV-induced LRTI (34 [43%] of 79 cases, P < 10⁻³) occurring more often in infants aged 1 to 12 months (P = 0.0002), with spring-fall seasonality. Viruses ECHO 11, 6, and 13 were the more frequently identified respiratory strains (24, 13, and 11%, respectively). The VP1 gene phylogenetic analysis showed the concomitant or successive circulation of genetically distinct EV respiratory strains (species A or B) during the same month or annual epidemic period. Our findings indicated that respiratory tract infections accounted for the 30% of EV-induced pediatric pathologies, contributing to LRTIs in 54% of these cases. Moreover, the concomitant or successive circulation of genetically distinct EV strains indicated the possibility of pediatric repeated respiratory infections within the same epidemic season.     

Online-Only Abstracts

The aims of our study were to analyse the risk factors for colonization by Extended-spectrum β-lactamases (ESBL)-producing Proteus mirabilis (ESBL-PM) in rehabilitation patients and to characterize the molecular features of these strains. The study was conducted in two rehabilitation centres located in Rome, Italy (Fondazione Santa Lucia IRCCS (FSL)), and Tel-Aviv, Israel (Tel-Aviv Sourasky Medical Center (TASMC)). Carriage of ESBL-PM was surveyed by rectal swabs. Strain typing was performed by pulsed-field gel electrophoresis (PFGE). Identification of ESBL genes was done by PCR and sequencing. Patients admitted to the same institutions without ESBL carriage were included as controls. The study group included 70 and 41 patients from FSL and TASMC, respectively. In FSL, the multivariate analysis identified severe acute brain injury (OR = 15, 95% CI = 3.2–69.5, p 0.001), decubitus ulcer (OR = 3.5, 95% CI = 1.2–9.8, p 0.018) and recent treatment with quinolones (OR = 5.7, 95% CI = 1.07–30.1, p 0.042) as independent risk factors. ESBL-PM carriers stayed longer in the hospital on average and were less likely to be discharged home. No significant risk factor was identified in TASMC. There were no similarities in PFGE types or ESBL genes between the ESBL-PM isolates from the two institutions. In both hospitals, a variety of PFGE types existed but a single ESBL type predominated, namely TEM-92 in FSL (n = 64/70; 91%) and CTX-M-2 in TASMC (n = 37/41; 90%). A new TEM ESBL variant, TEM-177 was identified in FSL. The clonal diversity and the predominance of a single ESBL type suggested that horizontal gene transfer played an important role in dissemination of resistance. The development of a population analysis tool that would allow tracing deeper genetic relationships is required.

The critical influence of the intermediate category on interpretation errors in revised EUCAST and CLSI antimicrobial susceptibility testing guidelines

M. Hombach¹, E. C. Böttger¹ and M. Roos²1) Institute of Medical Microbiology, University of Zurich and 2) Division of Biostatistics, Institute for Social and Preventive Medicine, University of Zurich, Zurich, SwitzerlandOriginal Submission: 22 August 2012; Revised Submission: 25 October 2012; Accepted: 25 October 2012Editor: F. AllerbergerArticle published online: 4 December 2012Clin Microbiol Infect 2013; 19: E59–E71

Abstract

Erroneous assignments of clinical isolates to the interpretative categories susceptible, intermediate and resistant can deprive a patient of successful antimicrobial therapy. The rate of major errors (ME) and very major errors (vME) is dependent on: (i) the precision/standard deviation (σ) of the antibiotic susceptibility testing (AST) method, (ii) the diameter distributions, (iii) clinical breakpoints, and (iv) the width of the intermediate zone. The European Committee on AST (EUCAST) has abandoned or decreased the intermediate zone for several drug/species combinations. This study focused on the effects of discontinuing the intermediate category on the rate of interpretation errors. In total, 10 341 non-duplicate clinical isolates were included in the study. For susceptibility testing the disc diffusion method was used. Error probabilities were calculated separately for diameter values flanking the interpretative category borders. Error probabilities were then applied to the actual numbers of clinical isolates investigated and expected rates of ME and vME were calculated. Applying EUCAST AST guidelines, significant rates of ME/vME were demonstrated for all drug/species combinations without an intermediate range. Virtually all ME/vME expected were eliminated in CLSI guidelines that retained an intermediate zone. If wild-type and resistant isolates are not clearly separated in susceptibility distributions, the retaining of an intermediate zone will decrease the number of ME and vME. An intermediate zone of 2–3 mm avoids almost all ME/vME for most species/drug combinations depending on diameter distributions. Laboratories should know their epidemiology settings to be able to detect problems of individual species/drug/clinical breakpoint combinations and take measures to improve precision of diameter measurements.

Bacteraemia due to OXA-48-carbapenemase-producing Enterobacteriaceae: a major clinical challenge

C. Navarro-San Francisco¹, M. Mora-Rillo¹, M. P. Romero-Gómez², F. Moreno-Ramos³, A. Rico-Nieto¹, G. Ruiz-Carrascoso², R. Gómez-Gil², J. R. Arribas-López¹, J. Mingorance² and J. R. Paño-Pardo¹1) Infectious Diseases and Clinical Microbiology Unit of the Internal Medicine and Microbiology Services, Hospital Universitario La Paz-IDIPAZ, 2) Microbiology Service, Hospital Universitario La Paz-IDIPAZ and 3) Pharmacy Service, Hospital Universitario La Paz-IDIPAZ, Madrid, SpainOriginal Submission: 16 August 2012; Revised Submission: 26 October 2012; Accepted: 26 October 2012Editor: F. AllerbergerArticle published online: 12 December 2012Clin Microbiol Infect 2013; 19: E72–E79

Abstract

Bacteraemia due to carbapenemase-producing Enterobacteriaceae is an emerging medical problem. Management of this entity is complicated by the difficulty in identifying resistance patterns and the limited therapeutic options. A cohort study was performed including all episodes of bloodstream infection due to OXA-48-producing Enterobacteriaceae (O48PE), occurring between July 2010 and April 2012. Data on predisposing factors, clinical presentation, therapy and outcome were collected from medical records. There were 40 cases of bacteraemia caused by O48PE, 35 Klebsiella pneumoniae and five Escherichia coli. Patients were elderly with significant comorbidities (57.5% underlying malignancy). Thirty-five cases (87.5%) were nosocomial, and five (12.5%) were healthcare-associated. Patients had frequently been exposed to antibiotics and to invasive procedures during hospitalization. The most common source of bacteraemia was the urinary tract followed by deep intra-abdominal surgical site infection. Clinical presentation was severe sepsis or shock in 18 cases (45%). Extended-spectrum β-lactamase production was detected in 92.5% of isolates. MIC₉₀ for ertapenem, imipenem and meropenem were 32, 16 and 16 mg/L, respectively. Most frequently preserved antibiotics were amikacin, colistin, tigecycline and fosfomycin. These antibiotics combined are the basis of targeted therapies, including carbapenem in selected cases. Median delay in starting clinically adequate and microbiologically appropriate treatment was 3 days. Crude mortality during admission and within 30 days from bacteraemia was 65% and 50%, respectively. Bloodstream infections caused by O48PE have a poor prognosis. Delay in diagnosis and in initiation of optimal antimicrobial therapy is frequent. Suspicion and rapid identification could contribute to improving outcomes.

Diagnosis of chronic brucellar meningitis and meningoencephalitis: the results of the Istanbul-2 study

H. Erdem¹, S. Kilic², B. Sener³, C. Acikel⁴, E. Alp⁵, M. Karahocagil⁶, F. Yetkin⁷, A. Inan⁸, V. Kecik-Bosnak⁹, H. C. Gul¹⁰, S. Tekin-Koruk¹ N. Ceran⁸, T. Demirdal¹², G. Yilmaz¹³, A. Ulu-Kilic⁵, B. Ceylan¹⁴, A. Dogan-Celik¹⁵, S. Nayman-Alpat¹⁶, R. Tekin¹⁷, A. Yalci¹⁸, V. Turhan¹, I. Karaoglan⁹, H. Yilmaz¹⁹, B. Mete²⁰, A. Batirel²¹, A. Ulcay¹, S. Dayan¹⁷, A. Seza Inal²², S. S. Ahmed⁵, Z. K. Tufan²³, A. Karakas¹⁰, B. Teker²⁴, M. Namiduru⁹, U. Savasci²⁵ and G. Pappas^26,271) Department of IDCM, Gulhane Haydarpasa Training Hospital, Istanbul,, 2) Public Health Institution of Turkey, Bacterial Zoonoses Reference Laboratory, Ankara, 3) Department of Microbiology and Clinical Microbiology, School of Medicine, Hacettepe University, Ankara, 4) Department of Public Health, Gulhane Medical Academy, Ankara, 5) Department of IDCM, School of Medicine, Erciyes University, Kayseri,, 6) Department of ID CM, School of Medicine, Yüzüncü Yil University, Van, 7) Department of IDCM, School of Medicine, Inonu University, Malatya, 8) Department of IDCM, Haydarpasa Numune, Training and Research Hospital, Istanbul, 9) Department of IDCM, School of Medicine, Gaziantep University, Gaziantep, 10) Department of IDCM, Gulhane School of Medicine, Ankara,, 11) Department of IDCM, School of Medicine, Harran University, Sanliurfa, 12) Department of IDCM, School of Medicine, Kocatepe University, Afyon,, 13) Department of IDCM, School of Medicine, Karadeniz Teknik University, Trabzon, 14) Department of IDCM, Medical Faculty, Bezmialem Vak?f University, Istanbul, 15) Department of IDCM, School of Medicine, Trakya University, Edirne, 16) Department of IDCM, School of Medicine, Osmangazi University, Eskisehir,, 17) Department of IDCM, School of Medicine, Dicle University, Diyarbakir, 18) Department of IDCM, School of Medicine, Ankara University, Ankara,, 19) Department of IDCM, School of Medicine, Ondokuz May?s University, Samsun, 20) Department of IDCM, Cerrahpasa School of Medicine, Istanbul University, Istanbul, 21) Department of IDCM, Kartal Dr Lutfi Kirdar Education and Research Hospital, Istanbul, 22) Department of IDCM, School of Medicine, Cukurova University, Adana, 23) Department of IDCM, Ankara Training and Research Hospital, Ankara, 24) Department of IDCM, Private Women‘s Hospital, Istanbul, 25) IDCM Service, Sarikamis Military Hospital, Kars, Turkey, 26) Institute of Continuing Medical Education of Ioannina, Ioannina, Greece and 27) Zoonoses Working Group, International Society of Chemotherapy, London, UKOriginal Submission: 17 June 2012; Revised Submission: 15 October 2012; Accepted: 31 October 2012Editor: M. DrancourtArticle published online: 4 December 2012Clin Microbiol Infect 2013; 19: E80–E86

Abstract

No detailed data exist in the literature on the accurate diagnosis of chronic brucellar meningitis or meningoencephalitis. A multicentre retrospective chart review was performed at 19 health centres to determine sensitivities of the diagnostic tests. This study included 177 patients. The mean values of CSF biochemical test results were as follows: CSF protein, 330.64 ± 493.28 mg/dL; CSF/ blood-glucose ratio, 0.35 ± 0.16; CSF sodium, 140.61 ± 8.14 mMt; CSF leucocyte count, 215.99 ± 306.87. The sensitivities of the tests were as follows: serum standard tube agglutination (STA), 94%; cerebrospinal fluid (CSF) STA, 78%; serum Rose Bengal test (RBT), 96%; CSF RBT, 71%; automated blood culture, 37%; automated CSF culture, 25%; conventional CSF culture, 9%. The clinician should use every possible means to diagnose chronic neurobrucellosis. The high seropositivitiy in brucellar blood tests must facilitate the use of blood serology. Although STA should be preferred over RBT in CSF in probable neurobrucellosis other than the acute form of the disease, RBT is not as weak as expected. Moreover, automated culture systems should be applied when CSF culture is needed.     

Broad-Range PCR-Electrospray Ionization Mass Spectrometry for Detection and Typing of Adenovirus and Other Opportunistic Viruses in Stem Cell Transplant Patients

Hematopoietic stem cell transplant patients are highly susceptible to viral infections. Follow-up after transplantation includes weekly screening using single, virus-specific real-time PCR tests, mainly for viruses in the families Herpesviridae and Adenoviridae that contribute to a high morbidity, especially in pediatric populations. The Abbott PLEX-ID platform combines broad-range PCR with electrospray ionization mass spectrometry to enable the simultaneous detection of multiple pathogens in a single assay. The Viral IC Spectrum assay detects human adenoviruses, viruses from the family Herpesviridae (herpes simplex virus 1 [HSV-1], HSV-2, cytomegalovirus [CMV], Epstein-Barr virus [EBV], varicella-zoster virus [VZV], and human herpesvirus 8 [HHV-8]), human enterovirus, polyomaviruses (BK and JC), and parvovirus B19. We evaluated the performance of the Viral IC Spectrum assay with samples from 16 adult and 36 pediatric stem cell transplant patients. The sensitivity of the Viral IC Spectrum assay compared to real-time PCR quantification using the adenovirus Rgene kit for the detection of adenovirus was 96.7% from plasma samples (n = 92) and 78% from stool samples (n = 100). No adenovirus was detected in samples from noninfected patients (n = 30). PLEX-ID species identification was perfectly concordant with species-specific real-time PCR assays. In plasma and stool samples, the level of amplified products measured by PLEX-ID and the quantity in copies/ml (r = 0.82 and 0.78, respectively) were correlated up to 6 log₁₀ copies/ml. In 67.4% of adenovirus-positive plasma samples, at least one other viral infection was detected; these included BK virus (n = 41), CMV (n = 30), EBV (n = 26), JC virus (n = 9), and HSV-1 (n = 6). The results of this study suggest that the Viral IC Spectrum assay performed on the PLEX-ID platform is reliable for adenovirus infection diagnosis in immunocompromised patients.     

Analysing myocardial tissue from explanted hearts of heart transplant recipients and multi-organ donors for the presence of parvovirus B19 DNA.

BACKGROUND: Parvovirus B19 (PVB19) is an erythrovirus causing diverse clinical manifestations ranging from asymptomatic or mild to more severe outcomes, dependent on the haematological and/or immunological status of the host. Reports of PVB19 infection as a causative agent of paediatric or adult inflammatory cardiac diseases, or of cardiac transplant rejection are rare. OBJECTIVES: To identify PVB19 and other cardiotropic viruses in the myocardium of heart transplant (HTx) recipients and multi-organ donors (MOD). Furthermore, to assess the prevalence of cardiotropic viral infection in inflammatory heart disease. STUDY DESIGN: Heart tissue samples from 110 explants were analysed for PVB19 using primers and a 5'-nuclease probe designed to amplify a 160-basepair PCR product from the VP1/NS1 gene region. Samples tested included those obtained from patients undergoing HTx or from MODs. The findings were correlated with clinical course, histologic analysis and serologic testing. Confirmation of the positive PCR-results was done by sequencing and in situ hybridisation. RESULTS: The new assay described here allows precise quantitation of viral load over 7 orders of magnitude (10(6) to 10(0)IU/assay). Measurable amounts of parvoviral genomes were detected in 4/56 (7%) explanted HTx-hearts and in 5/54 (9%) explanted MOD-hearts. CONCLUSIONS: The newly developed real-time PCR is a rapid, sensitive and specific method to detect PVB19 infection in heart tissue. It will be a useful tool to address important questions regarding viral infections transmitted by transplantation, acute infections, relapses and complications involving late or chronic rejection.     

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.     

The detection,treatment of parvovirus B19 infection induced anemia in solid organ transplants: A case series and literature review of 194 patients

BackgroundThere are no optimal diagnostic, treatment and post-infection surveillance strategies for parvovirus B19 infection in solid organ transplantation (SOT) recipients.MethodsWe conducted a retrospective review of all PVB19 infected cases confirmed by qPCR among SOT recipients at our institution over a 3-year period and reviewed the literature from 1990 to 2021.ResultsEight kidney and two heart transplant patients with refractory anemia had PVB19 infection. The viral DNA load in peripheral blood ranged from 2.62 × 10² to 8.31 × 10⁶ copies/mL. Two patients with the lowest PVB19 DNA load only reduced the use of immunosuppressants and anemia was relieved. Eight received intravenous immunoglobulin (IVIG) (ranging from 0.25 to 0.5 g/kg/day). The median time to anemia improvement (hemoglobulin > 100 g/L) was 16 days (8–70 days) after treatment. One patient had a PVB19 relapse and viral DNA load > 1.00 × 10⁸ copies/mL at diagnosis. A total of 86 studies involving 194 SOTs were screened from the literature, and the most common symptom was anemia and low reticulocyte count. PVB19 DNA was detected in all cases. Of that, 91.4% of cases received IVIG, 53.8% received IVIG and immunosuppression reduction, 6.5% of cases showed reduced immunosuppression without IVIG, and 2.1% did not receive any special treatment. The recurrence rate was 17.5%.ConclusionPVB19 infection is a cause of anemia after SOT, and treatment mainly relies on IVIG and/or immunosuppression reduction.     

Laboratory Evaluation of the Liat HIV Quant (IQuum) Whole-Blood and Plasma HIV-1 Viral Load Assays for Point-of-Care Testing in South Africa

Point-of-care (POC) HIV viral load (VL) testing offers the potential to reduce turnaround times for antiretroviral therapy monitoring, offer near-patient acute HIV diagnosis in adults, extend existing centralized VL services, screen women in labor, and prompt pediatrics to early treatment. The Liat HIV Quant plasma and whole-blood assays, prerelease version, were evaluated in South Africa. The precision, accuracy, linearity, and agreement of the Liat HIV Quant whole-blood and plasma assays were compared to those of reference technologies (Roche CAP CTMv2.0 and Abbott RealTime HIV-1) on an HIV verification plasma panel (n = 42) and HIV clinical specimens (n = 163). HIV Quant plasma assay showed good performance, with a 2.7% similarity coefficient of variation (CV) compared to the Abbott assay and a 1.8% similarity CV compared to the Roche test on the verification panel, and 100% specificity. HIV Quant plasma had substantial agreement (p_c [concordance correlation] = 0.96) with Roche on clinical specimens and increased variability (p_c = 0.73) in the range of <3.0 log copies/ml range with the HIV Quant whole-blood assay. HIV Quant plasma assay had good linearity (2.0 to 5.0 log copies/ml; R² = 0.99). Clinical sensitivity at a viral load of 1,000 copies/ml of the HIV Quant plasma and whole-blood assays compared to that of the Roche assay (n = 94) was 100% (confidence interval [CI], 95.3% to 100%). The specificity of HIV Quant plasma was 88.2% (CI, 63.6% to 98.5%), and that for whole blood was 41.2% (CI, 18.4% to 67.1%). No virological failure (downward misclassification) was missed. Liat HIV Quant plasma assay can be interchanged with existing VL technology in South Africa. Liat HIV Quant whole-blood assay would be advantageous for POC early infant diagnosis at birth and adult adherence monitoring and needs to be evaluated further in this clinical context. LIAT cartridges currently require cold storage, but the technology is user-friendly and robust. Clinical cost and implementation modeling is required.     

Comparison of the GenMark Diagnostics eSensor Respiratory Viral Panel to Real-Time PCR for Detection of Respiratory Viruses in Children

A novel eSensor respiratory viral panel (eSensor RVP) multiplexed nucleic acid amplification test (GenMark Diagnostics, Inc., Carlsbad, CA) was compared to laboratory-developed real-time PCR assays for the detection of various respiratory viruses. A total of 250 frozen archived pediatric respiratory specimens previously characterized as either negative or positive for one or more viruses by real-time PCR were examined using the eSensor RVP. Overall agreement between the eSensor RVP and corresponding real-time PCR assays for shared analytes was 99.2% (kappa = 0.96 [95% confidence interval {CI}, 0.94 to 0.98]). The combined positive percent agreement was 95.4% (95% CI, 92.5 to 97.3); the negative percent agreement was 99.7% (95% CI, 99.4 to 99.8). The mean real-time PCR threshold cycle (C_T) value for specimens with discordant results was 39.73 (95% CI, 38.03 to 41.43). Detection of coinfections and correct identification of influenza A virus subtypes were comparable between methods. Of note, the eSensor RVP rhinovirus assay was found to be more sensitive and specific than the corresponding rhinovirus real-time PCR. In contrast, the eSensor RVP adenovirus B, C, and E assays demonstrated some cross-reactivity when tested against known adenovirus serotypes representing groups A through F. The eSensor RVP is robust and relatively easy to perform, it involves a unique biosensor technology for target detection, and its multiplexed design allows for efficient and simultaneous interrogation of a single specimen for multiple viruses. Potential drawbacks include a slower turnaround time and the need to manipulate amplified product during the protocol, increasing the possibility of contamination.     

Type-Specific Detection of 30 Oncogenic Human Papillomaviruses by Genotyping both E6 and L1 Genes

Human papillomavirus (HPV) is the principal cause of invasive cervical cancer and benign genital lesions. There are currently 30 HPV types linked to cervical cancer. HPV infection also leads to other types of cancer. We developed a 61-plex analysis of these 30 HPV types by examining two genes, E6 and L1, using MassARRAY matrix-assisted laser desorption ionization–time of flight mass spectrometry (MALDI-TOF MS) (PCR-MS). Two hundred samples from homosexual males (HM) were screened by PCR-MS and MY09/MY11 primer set-mediated PCR (MY-PCR) followed by sequencing. One hundred thirty-five formalin-fixed, paraffin-embedded (FFPE) cervical cancer samples were also analyzed by PCR-MS, and results were compared to those of the commercially available GenoArray (GA) assay. One or more HPV types were identified in 64.5% (129/200) of the samples from HM. Comprising all 30 HPV types, PCR-MS detected 51.9% (67/129) of samples with multiple HPV types, whereas MY-PCR detected only one single HPV type in these samples. All PCR-MS results were confirmed by MY-PCR. In the cervical cancer samples, PCR-MS and GA detected 97% (131/135) and 90.4% (122/135) of HPV-positive samples, respectively. PCR-MS and GA results were fully concordant for 122 positive and 4 negative samples. The sequencing results for the 9 samples that tested negative by GA were completely concordant with the positive PCR-MS results. Multiple HPV types were identified in 25.2% (34/135) and 55.6% (75/135) of the cervical cancer samples by GA and PCR-MS, respectively, and results were confirmed by sequencing. The new assay allows the genotyping of >1,000 samples per day. It provides a good alternative to current methods, especially for large-scale investigations of multiple HPV infections and degraded FFPE samples.     

Development and Evaluation of a Next-Generation Digital PCR Diagnostic Assay for Ocular Chlamydia trachomatis Infections

Droplet digital PCR (ddPCR) is an emulsion PCR process that performs absolute quantitation of nucleic acids. We developed a ddPCR assay for Chlamydia trachomatis infections and found it to be accurate and precise. Using PCR mixtures containing plasmids engineered to include the PCR target sequences, we were able to quantify with a dynamic range between 0.07 and 3,160 targets/μl (r² = 0.9927) with >95% confidence. Using 1,509 clinical conjunctival swab samples from a population in which trachoma is endemic in Guinea Bissau, we evaluated the specificity and sensitivity of the quantitative ddPCR assay in diagnosing ocular C. trachomatis infections by comparing the performances of ddPCR and the Roche Amplicor CT/NG test. We defined ddPCR tests as positive when we had ≥95% confidence in a nonzero estimate of target load. The sensitivity of ddPCR against Amplicor was 73.3% (95% confidence interval [CI], 67.9 to 78.7%), and specificity was 99.1% (95% CI, 98.6 to 99.6%). Negative and positive predictive values were 94.6% (95% CI, 93.4 to 95.8%) and 94.5% (95% CI, 91.3 to 97.7%), respectively. Based on Amplicor CT/NG testing, the estimated population prevalence of C. trachomatis ocular infection was ∼17.5%. Receiver-operator curve analysis was used to select critical cutoff values for use in clinical settings in which a balance between higher sensitivity and specificity is required. We concluded that ddPCR is an effective diagnostic technology suitable for both research and clinical use in diagnosing ocular C. trachomatis infections.     

Detection and enumeration of transformation-defective strains of avian sarcoma virus with molecular hybridization.

Serial propagation of avian sarcoma viruses generates deletions in the viral gene responsible for cellular transformation (src). We have devised an assay for these deletion mutants which utilizes molecular hybridization and exploits the availability of DNA (cDNA_sarc) complementary to the nucleotide sequences affected by the deletion in src. Our procedure is also applicable to deletions in other viral genes and offers several advantages over conventional bioassays for the deletion mutants; moreover, it can be used to detect deletions in virus-specific intracellular nucleic acids. In order to illustrate the utility of the assay, we demonstrate that all 20 copies of the proviral DNA for avian sarcoma viruses in XC cells contain src, and we show that single avian cells can contain functioning proviruses for both avian sarcoma virus and a congenic deletion mutant. It should now be possible to use molecular hybridization to study the mechanism by which deletions in src are generated.