Virological assessment of hospitalized patients with COVID-2019.

Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.

Experimental Assessment of Zika Virus Mechanical Transmission by Aedes Aegypti.

The pandemic emergence of several mosquito-borne viruses highlights the need to understand the different ways in which they can be transmitted by vectors to human hosts. In this study, we evaluated the propensity of Aedes aegypti to transmit mechanically Zika virus (ZIKV) using an experimental design. Mosquitoes were allowed to feed on ZIKV-infected blood and were then rapidly transferred to feed on ZIKV-free blood until they finished their meal. The uninfected blood meals, the mosquito abdomens, as well as the mouthparts dissected from fully and partially engorged mosquitoes were analyzed using RT-qPCR and/or virus titration. All the fully engorged mosquito abdomens were ZIKV-infected, whereas their mouthparts were all ZIKV-negative. Nonetheless, one of the partially engorged mosquitoes carried infectious particles on mouthparts. No infectious virus was found in the receiver blood meals, while viral RNA was detected in 9% of the samples (2/22). Thus, mechanical transmission of ZIKV may sporadically occur via Ae. aegypti bite. However, as the number of virions detected on mouthparts (2 particles) is not sufficient to induce infection in a naïve host, our results indicate that mechanical transmission does not impact ZIKV epidemiology.

Evaluation of rapid and sensitive DNA extraction methods for detection of cytomegalovirus in dried blood spots.

BACKGROUND: Dried blood spots (DBS), collected universally from newborns in the U.S., could be used as a matrix for the detection of cytomegalovirus (CMV) infection in infants. However, sensitivity to detect CMV in DBS as compared to saliva and urine is variable across studies largely due to the DNA extraction method. Thermal shock, a widely used DNA extraction method, is highly sensitive for the detection of CMV in DBS, however, the processing time required is not practical for high-throughput testing. OBJECTIVE: To determine if rapid and cost-effective DNA extraction methods amenable to newborn screening (NBS) could achieve the same sensitivity as the thermal shock method. STUDY DESIGN: DBS were prepared from CMV positive blood samples from 20 organ transplant recipients. Three DNA extraction methods were compared for relative yield and sensitivity of detection of CMV DNA: thermal shock, KOH Tris buffer, and DNA Extract All. CMV DNA was detected by real-time quantitative polymerase chain reaction (qPCR). RESULTS: The KOH Tris and DNA Extract All methods gave higher yields and sensitivity of CMV detection in DBS than thermal shock, which were significantly greater when viral loads were ≤ 10,000 copies/ml blood. Both methods gave faster turnaround times than thermal shock and would be better suited for NBS. CONCLUSIONS: The choice of DNA extraction method greatly influences the ability to detect low levels of CMV DNA in DBS. Moreover, development of highly sensitive yet rapid methods for CMV detection could help facilitate future newborn screening of CMV in DBS.

Assessment of blood enterovirus PCR testing in paediatric populations with fever without source, sepsis-like disease, or suspected meningitis: a prospective, multicentre, observational cohort study.

BACKGROUND: Enteroviruses are the most frequent cause of acute meningitis and are seen increasingly in sepsis-like disease and fever without source in the paediatric population. Detection of enterovirus in cerebrospinal fluid (CSF) specimens by PCR is the gold standard diagnostic test. Our aim was to assess a method of detecting enterovirus in blood specimens by PCR. METHODS: We did a prospective, multicentre, observational study at 35 French paediatric and emergency departments in 16 hospitals. We recruited newborn babies (aged ≤28 days) and infants (aged >28 days to ≤2 years) with fever without source, sepsis-like disease, or suspected meningitis, and children (aged >2 years to ≤16 years) with suspected meningitis, who were admitted to a participating hospital. We used a standardised form to obtain demographic, clinical, and laboratory data, which were anonymised. Enterovirus PCR testing was done in blood and CSF specimens. FINDINGS: Between June 1, 2015, and Oct 31, 2015, and between June 1, 2016, and Oct 31, 2016, we enrolled 822 patients, of whom 672 had enterovirus PCR testing done in blood and CSF specimens. Enterovirus was detected in 317 (47%) patients in either blood or CSF, or both (71 newborn babies, 83 infants, and 163 children). Detection of enterovirus was more frequent in blood samples than in CSF specimens of newborn babies (70 [99%] of 71 vs 62 [87%] of 71; p=0·011) and infants (76 [92%] of 83 vs 62 [75%] of 83; p=0·008), and was less frequent in blood samples than in CSF specimens of children (90 [55%] of 163 vs 148 [91%] of 163; p<0·0001). Detection of enterovirus was more frequent in blood samples than in CSF specimens of infants aged 2 years or younger with fever without source (55 [100%] of 55 vs 41 [75%] of 55; p=0·0002) or with sepsis-like disease (16 [100%] of 16 vs nine [56%] of 16; p=0·008). Detection of enterovirus was less frequent in blood than in CSF of patients with suspected meningitis (165 [67%] of 246 vs 222 [90%] of 246; p<0·0001). INTERPRETATION: Testing for enterovirus in blood by PCR should be an integral part of clinical practice guidelines for infants aged 2 years or younger. This testing could decrease the length of hospital stay and reduce exposure to antibiotics for low-risk patients admitted to the emergency department with febrile illness. FUNDING: University Hospital Clermont-Ferrand.

Investigational Testing for Zika Virus among U.S. Blood Donors.

BACKGROUND: Because of the potential severe clinical consequences of Zika virus (ZIKV) infection, the large numbers of asymptomatic travelers returning from ZIKV-active areas, the detection of ZIKV nucleic acid in blood, and reports of transmission of ZIKV through transfusion, in 2016 the Food and Drug Administration released recommendations for individual-unit nucleic acid testing to minimize the risk of transmission of ZIKV through blood transfusions. METHODS: The American Red Cross implemented investigational screening of donated blood for ZIKV RNA by means of transcription-mediated amplification (TMA). Confirmatory testing of reactive donations involved repeat TMA, TMA testing in exploratory minipools, real-time reverse-transcriptase polymerase chain reaction, IgM serologic testing, and red-cell TMA. Viral loads in plasma and red cells were estimated by means of end-point TMA. The costs of interdicting a donation that was confirmed to be positive were calculated for the 15-month period between June 2016 and September 2017. RESULTS: Of the 4,325,889 donations that were screened, 393,713 (9%) were initially tested in 24,611 minipools, and no reactive donations were found. Of the 3,932,176 donations that were subsequently tested individually, 160 were initially reactive and 9 were confirmed positive (a 1:480,654 confirmed-positive rate overall; positive predictive value, 5.6%; specificity, 99.997%). Six (67%) of the confirmed-positive donations were reactive on repeat TMA, of which 4 were IgM-negative; of these 4, all 3 that could be tested were reactive on minipool TMA. Two confirmed-positive donors had infections that had been transmitted locally (in Florida), 6 had traveled to ZIKV-active areas, and 1 had received an experimental ZIKV vaccine. ZIKV RNA levels in red cells ranged from 40 to 800,000 copies per milliliter and were detected up to 154 days after donation, as compared with 80 days of detection in plasma at levels of 12 to 20,000 copies per milliliter. On the basis of industry-reported costs of testing and the yield of the tests in our study, the cost of identifying 8 mosquito-borne ZIKV infections through individual-unit nucleic acid testing was $5.3 million per ZIKV RNA-positive donation. CONCLUSIONS: Screening of U.S. blood donations for ZIKV by individual-donation TMA was costly and had a low yield. Among the 9 confirmed ZIKV-positive donations, only 4 were IgM-negative; of these donations, all 3 that were tested were reactive on minipool TMA. (Funded by the American Red Cross and Grifols Diagnostic Solutions.).

Seroprevalence, risk associations, and cost analysis of screening for viral infections among patients of cataract surgery.

PURPOSE: The purpose of this study was to estimate the prevalence of blood-borne viral infections (triple H: HBV-hepatitis B virus, HCV-hepatitis C virus, and HIV-human immunodeficiency virus) among cataract patients, sought possible risk associations and discuss feasibility of universal preoperative screening. METHODS: This prospective, cross-sectional study enrolled consecutive patients of senile cataract. They were screened by immunoassay-based rapid diagnostic card tests for blood-borne viral infections. Positive cases were confirmed with confirmatory ELISA tests. Seropositive patients were enquired about the exposure to possible risk associations for acquiring these infections. Cost of card test per patient was calculated. RESULTS: The prevalence of seropositivity for triple H viral infections (HBV, HCV, and HIV) among patients of senile cataract was 5.9% (95% confidence interval [CI]: 5.3-6.6), and HCV was most common viral infection. The dental extraction was most common (54%; 95% CI:48-60) possible risk association. The total cost of primary screening per patient for triple H infections(HBV, HCV, and HIV) was $0.93. CONCLUSION: The prevalence of blood-borne viral infection among cataract patients is high in this area. Awareness of the prevalence of blood-borne viral infections in service area, along with knowledge of rate of accidental exposure and risk of transmission would help to understand cost-effectiveness of universal preoperative screening before cataract surgery.

Assessment of semi-automated nucleic acid testing programme in a Regional Blood Transfusion Centre.

BACKGROUND: Detection of human immunodeficiency virus type-1 (HIV-1), hepatitis-C (HCV) and hepatitis-B virus (HBV) in the blood donors is crucial. An efficient form of detection is nucleic acid testing (NAT) in blood screening. We assessed the suitability of commercial NAT testing in a developing country, focusing on the Altona RealStar assay and the method of Sacace Biotechnologies. METHODS: We have standardised and validated commercially available NAT kits with a semi-automated system for detection of HBV, HCV and HIV-1 in blood donations. The MP-NAT (mini-pool) assay consists of pooling of sample, virus extraction, amplification and detection with commercially available NAT kits. An internal control (IC) is incorporated in the assay to monitor the extraction, target amplification and detection process. RESULTS: The sensitivity of the Altona RealStar assay at 10-MP for each viral target was evaluated, HBV showed amplification in all diluted positive samples of 100, 50, 25, 10 and 5 IU/ml. HIV and HCV infected samples showed amplification in all diluted positive samples of 500, 100, 50 and 30 IU/ml. For HIV, out of six diluted samples of 30 IU/ml, five were amplified. A total of 14,170 seronegative blood samples were tested by RealStar PCR kit in 10-MP and 6 (0.042%) samples/pools were positive. A total of 65,362 seronegative blood donations were also tested by kits of Sacace Biotechnologies, in 10-MP and 45 (0.075%) pools were positive. The prevalence of combined NAT yield cases among routine donors was 1 in 1559 donations tested for all the 3 viruses. CONCLUSION: The semi-automated combined system for NAT screening assays is robust, sensitive, reproducible, and this gives an additional layer of safety with affordable cost.

Early antiretroviral therapy initiation: access and equity of viral load testing for HIV treatment monitoring.

Scaling up access to HIV viral load testing for individuals undergoing antiretroviral therapy in low-resource settings is a global health priority, as emphasised by research showing the benefits of suppressed viral load for the individual and the whole population. Historically, large-scale diagnostic test implementation has been slow and incomplete because of service delivery and other challenges. Building on lessons from the past, in this Personal View we propose a new framework to accelerate viral load scale-up and ensure equitable access to this essential test. The framework includes the following steps: (1) ensuring adequate financial investment in scaling up this test; (2) achieving pricing agreements and consolidating procurement to lower prices of the test; (3) strengthening functional tiered laboratory networks and systems to expand access to reliable, high-quality testing across countries; (4) strengthening national leadership, with prioritisation of laboratory services; and (5) demand creation and uptake of test results by clinicians, nurses, and patients, which will be vital in ensuring viral load tests are appropriately used to improve the quality of care. The use of dried blood spots to stabilise and ship samples from clinics to laboratories, and the use of point-of-care diagnostic tests, will also be important for ensuring access, especially in settings with reduced laboratory capacity. For countries that have just started to scale up viral load testing, lessons can be learnt from countries such as Botswana, Brazil, South Africa, and Thailand, which have already established viral load programmes. This framework might be useful for guiding the implementation of viral load with the aim of achieving the new global HIV 90-90-90 goals by 2020.

Sensitivity and specificity of dried blood spots for HIV-1 viral load quantification: A laboratory assessment of 3 commercial assays.

The use of dried blood spots (DBS) instead of plasma as a specimen type for HIV-1 viral load (VL) testing facilitates the decentralization of specimen collection and can increase access to VL testing in resource-limited settings. The performance of DBS for VL testing is lower, however, when compared to the gold standard sample type plasma. In this diagnostic accuracy study, we evaluated 3 VL assays with DBS.Participants were recruited between August 2012 and April 2015. Both plasma and DBS specimens were prepared and tested for HIV-1 VL with the Roche CAP/CTM HIV-1 test v2.0, the Abbott RealTime HIV-1, and the bioMérieux NucliSENS EasyQ HIV-1 v2.0. Sensitivity and specificity to detect treatment failure at a threshold of 1000 cps/mL with DBS were determined.A total of 272 HIV-positive patients and 51 HIV-negative people were recruited in the study. The mean difference or bias between plasma and DBS VL was <0.5 log cps/mL with all 3 assays but >25% of the specimens differed by >0.5 log cps/mL.All 3 assays had comparable sensitivities around 80% and specificities around 90%. Upward misclassification rates were around 10%, but downward misclassification rates ranged from 20.3% to 23.6%. Differences in between assays were not statistically significant (P > 0.1).The 3 VL assays evaluated had suboptimal performance with DBS but still performed better than immunological or clinical monitoring. Even after the introduction of the much-anticipated point-of-care VL devices, it is expected that DBS will remain important as a complementary option for supporting access to VL monitoring, particularly in rural, resource-limited settings. Manufacturers should accelerate efforts to develop more reliable, sensitive and specific methods to test VL on DBS specimens.

Rapid, Low-Cost Detection of Zika Virus Using Programmable Biomolecular Components.

The recent Zika virus outbreak highlights the need for low-cost diagnostics that can be rapidly developed for distribution and use in pandemic regions. Here, we report a pipeline for the rapid design, assembly, and validation of cell-free, paper-based sensors for the detection of the Zika virus RNA genome. By linking isothermal RNA amplification to toehold switch RNA sensors, we detect clinically relevant concentrations of Zika virus sequences and demonstrate specificity against closely related Dengue virus sequences. When coupled with a novel CRISPR/Cas9-based module, our sensors can discriminate between viral strains with single-base resolution. We successfully demonstrate a simple, field-ready sample-processing workflow and detect Zika virus from the plasma of a viremic macaque. Our freeze-dried biomolecular platform resolves important practical limitations to the deployment of molecular diagnostics in the field and demonstrates how synthetic biology can be used to develop diagnostic tools for confronting global health crises. PAPERCLIP.

Incorporating HIV/hepatitis B virus/hepatitis C virus combined testing into routine blood tests in nine UK Emergency Departments: the "Going Viral" campaign.

OBJECTIVES: Routine HIV screening is recommended in those UK hospitals and primary care settings where the HIV prevalence is > 0.2%. For hepatitis B virus (HBV) and hepatitis C virus (HCV), however, testing is targeted at at-risk groups. We investigated the prevalence of these blood-borne viruses (BBVs) during a routine testing pilot in UK Emergency Departments (EDs). METHODS: During the "Going Viral" campaign (13-19 October 2014), nine UK EDs in areas of high HIV prevalence offered routine tests for HIV, HBV and HCV to adults having blood taken as part of routine care. Patients who tested positive were linked to care. RESULTS: A total of 7807 patients had blood taken during their ED visit; of these, 2118 (27%) were tested for BBVs (range 9-65%). Seventy-one BBV tests were positive (3.4%) with 32 (45.1%) new diagnoses. There were 39 HCV infections (15 newly diagnosed), 17 HIV infections (six newly diagnosed), and 15 HBV infections (11 newly diagnosed). Those aged 25-54 years had the highest prevalence: 2.46% for HCV, 1.36% for HIV and 1.09% for HBV. Assuming the cost per diagnosis is £7, the cost per new case detected would be £988 for HCV, £1351 for HBV and £2478 for HIV. CONCLUSIONS: In the first study in the UK to report prospectively on BBV prevalence in the ED, we identified a high number of new viral hepatitis diagnoses, especially hepatitis C, in addition to the HIV diagnoses. Testing for HIV alone would have missed 54 viral hepatitis diagnoses (26 new), supporting further evaluation of routine BBV testing in UK EDs.

Design and implementation of an external quality assessment program for HIV viral load measurements using dried blood spots.

An external quality assurance program was developed for HIV-1 RNA viral load measurements taken from dried blood spots using a reference panel and field-collected specimens. The program demonstrated that accurate and reproducible quantitation can be obtained from field-collected specimens. Residual proviral DNA may confound interpretation in virologically suppressed subjects.

Characterization of small ruminant lentivirus A4 subtype isolates and assessment of their pathogenic potential in naturally infected goats.

BACKGROUND: Small ruminant lentiviruses escaping efficient serological detection are still circulating in Swiss goats in spite of a long eradication campaign that essentially eliminated clinical cases of caprine arthritis encephalitis in the country. This strongly suggests that the circulating viruses are avirulent for goats.To test this hypothesis, we isolated circulating viruses from naturally infected animals and tested the in vitro and in vivo characteristics of these field isolates. METHODS: Viruses were isolated from primary macrophage cultures. The presence of lentiviruses in the culture supernatants was monitored by reverse transcriptase assay. Isolates were passaged in different cells and their cytopathogenic effects monitored by microscopy. Proviral load was quantified by real-time PCR using customized primer and probes. Statistical analysis comprised Analysis of Variance and Bonferroni Multiple Comparison Test. RESULTS: The isolated viruses belonged to the small ruminant lentiviruses A4 subtype that appears to be prominent in Switzerland. The 4 isolates replicated very efficiently in macrophages, displaying heterogeneous phenotypes, with two isolates showing a pronounced cytopathogenicity for these cells. By contrast, all 4 isolates had a poor replication capacity in goat and sheep fibroblasts. The proviral loads in the peripheral blood and, in particular, in the mammary gland were surprisingly high compared to previous observations. Nevertheless, these viruses appear to be of low virulence for goats except for the mammary gland were histopathological changes were observed. CONCLUSIONS: Small ruminant lentiviruses continue to circulate in Switzerland despite a long and expensive caprine arthritis encephalitis virus eradication campaign. We isolated 4 of these lentiviruses and confirmed their phylogenetic association with the prominent A4 subtype. The pathological and histopathological analysis of the infected animals supported the hypothesis that these A4 viruses are of low pathogenicity for goats, with, however, a caveat about the potentially detrimental effects on the mammary gland. Moreover, the high proviral load detected indicates that the immune system of the animals cannot control the infection and this, combined with the phenotypic plasticity observed in vitro, strongly argues in favour of a continuous and precise monitoring of these SRLV to avoid the risk of jeopardizing a long eradication campaign.

Dried-blood spots: a cost-effective field method for the detection of Chikungunya virus circulation in remote areas.

BACKGROUND: In 2005, there were outbreaks of febrile polyarthritis due to Chikungunya virus (CHIKV) in the Comoros Islands. CHIKV then spread to other islands in the Indian Ocean: La Réunion, Mauritius, Seychelles and Madagascar. These outbreaks revealed the lack of surveillance and preparedness of Madagascar and other countries. Thus, it was decided in 2007 to establish a syndrome-based surveillance network to monitor dengue-like illness. OBJECTIVE: This study aims to evaluate the use of capillary blood samples blotted on filter papers for molecular diagnosis of CHIKV infection. Venous blood samples can be difficult to obtain and the shipment of serum in appropriate temperature conditions is too costly for most developing countries. METHODOLOGY AND PRINCIPAL FINDINGS: Venous blood and dried-blood blotted on filter paper (DBFP) were collected during the last CHIKV outbreak in Madagascar (2010) and as part of our routine surveillance of dengue-like illness. All samples were tested by real-time RT-PCR and results with serum and DBFP samples were compared for each patient. The sensitivity and specificity of tests performed with DBFP, relative to those with venous samples (defined as 100%) were 93.1% (95% CI:[84.7-97.7]) and 94.4% (95% CI:[88.3-97.7]), respectively. The Kappa coefficient 0.87 (95% CI:[0.80-0.94]) was excellent. CONCLUSION: This study shows that DBFP specimens can be used as a cost-effective alternative sampling method for the surveillance and monitoring of CHIKV circulation and emergence in developing countries, and probably also for other arboviruses. The loss of sensitivity is insignificant and involved a very small number of patients, all with low viral loads. Whether viruses can be isolated from dried blood spots remains to be determined.

Multifunctional electrochemical aptasensor for aptamer clones screening, virus quantitation in blood and viability assessment.

A novel attempt was made to develop a disposable multifunctional sensor for analysis of vaccinia virus (VACV), a promising oncolytic agent that can replicate in and kill tumor cells. Briefly, we developed aptamers specific to VACV that were negatively selected against human serum as well as human and mouse blood to be further utilized for viral analysis directly in serum and blood. In addition, the aptamers were negatively selected against heat-inactivated VACV to enable them to distinguish between viable and nonviable virus particles. The selected aptamers were integrated onto an electrochemical aptasensor to perform multiple functions, including quantification of VACV, viability assessment of the virus, and estimation of the binding affinity between the virus and the developed aptamers. The aptasensor was fabricated by self-assembling a hybrid of a thiolated ssDNA primer and a VACV-specific aptamer onto a gold nanoparticles modified screen-printed carbon electrode (GNPs-SPCE). Square wave voltammetry was employed to quantify VACV in serum and blood within the range of 150-900 PFU, with a detection limit of 60 PFU in 30 µL. According to the electrochemical affinity measurements, three virus specific aptamer clones, V-2, V-5, and V-9 exhibited the highest affinity to VACV. Furthermore, flow cytometry was employed to estimate the dissociation constants of the clones which were found to be 26.3, 40.9, and 24.7 nM, respectively. Finally, the developed aptasensor was able to distinguish between the intact virus and the heat-inactivated virus thanks to the tailored selectivity of the aptamers that was achieved via negative selection.

Challenges and opportunities for the implementation of virological testing in resource-limited settings.

Though the advantages of routine virological monitoring for patients on anti-retroviral therapy have been established, cost and complexity limit its full implementation. Monitoring is important for diagnosing virological failure early on, before the development of drug resistance mutations, and to trigger early adherence interventions. Simple and cost-effective viral load tests that facilitate simplification and decentralization of testing and strategies, such as the use of dried blood spots and pooled sample testing, which further aid simplification, are becoming available. In addition, replacing immunological monitoring with virological monitoring in non-viremic patients in a phased manner will reduce the costs associated with dual immuno-virological monitoring. Going forward, the simplification of testing paired with price reducing strategies that will allow for healthy competition between multiple manufacturers will enable the implementation of viral load testing in resource-poor settings. It is important that future HIV and AIDS treatment guidelines provide clear recommendations for routine virological monitoring and that governments and donors fund the implementation of accurate and operationally proven testing platforms in a comprehensive manner.

The use of a mobile laboratory unit in support of patient management and epidemiological surveillance during the 2005 Marburg Outbreak in Angola.

BACKGROUND: Marburg virus (MARV), a zoonotic pathogen causing severe hemorrhagic fever in man, has emerged in Angola resulting in the largest outbreak of Marburg hemorrhagic fever (MHF) with the highest case fatality rate to date. METHODOLOGY/PRINCIPAL FINDINGS: A mobile laboratory unit (MLU) was deployed as part of the World Health Organization outbreak response. Utilizing quantitative real-time PCR assays, this laboratory provided specific MARV diagnostics in Uige, the epicentre of the outbreak. The MLU operated over a period of 88 days and tested 620 specimens from 388 individuals. Specimens included mainly oral swabs and EDTA blood. Following establishing on site, the MLU operation allowed a diagnostic response in <4 hours from sample receiving. Most cases were found among females in the child-bearing age and in children less than five years of age. The outbreak had a high number of paediatric cases and breastfeeding may have been a factor in MARV transmission as indicated by the epidemiology and MARV positive breast milk specimens. Oral swabs were a useful alternative specimen source to whole blood/serum allowing testing of patients in circumstances of resistance to invasive procedures but limited diagnostic testing to molecular approaches. There was a high concordance in test results between the MLU and the reference laboratory in Luanda operated by the US Centers for Disease Control and Prevention. CONCLUSIONS/SIGNIFICANCE: The MLU was an important outbreak response asset providing support in patient management and epidemiological surveillance. Field laboratory capacity should be expanded and made an essential part of any future outbreak investigation.

The harder you look, the more you find: Catheter-associated bloodstream infection surveillance variability.

BACKGROUND: Catheter-related bloodstream infections are an important quality performance measure and remain a significant source of added morbidity, mortality, and medical costs. OBJECTIVE: Our objectives were to assess variability in catheter-associated bloodstream infections (CA-BSI) surveillance practices, management, and attitudes/beliefs in pediatric intensive care units (PICUs) and to determine whether any correlation exists between surveillance variation and CA-BSI rates. METHODS: We used a survey of 5 health care professions at multiple institutions. RESULTS: One hundred forty-six respondents from 5 professions in 16 PICUs completed surveys with a response rate of 40%. All 10 (100%) infection control departments reported inclusion or exclusion of central line types inconsistent with the Centers for Disease Control and Prevention CA-BSI definition, 5 (50%) calculated line-days inconsistently, and only 5 (50%) used a strict, written policy for classifying BSIs. Infection control departments report substantial variation in methods, timing, and resources used to screen and adjudicate BSI cases. Greater than 80% of centers report having a formal, written policy about obtaining blood cultures, although less than 80% of these address obtaining samples from patients with central venous lines, and any such policies are reportedly followed less than half of the time. Substantial variation exists in blood culturing practices, such as temperature thresholds, preemptive antipyretics, and blood sampling (volumes, number, sites, frequencies). A surveillance aggressiveness score was devised to quantify practices likely to increase identification of bloodstream infections, and there was a significant correlation between the surveillance aggressiveness score and CA-BSI rates (r = 0.60, P = .034). In assessing attitudes and beliefs, there was much greater confidence in the validity of CA-BSI as an internal/historical benchmark than as an external/peer benchmark, and the factor most commonly believed to contribute to CA-BSI occurrences was patient risk factors, not central line maintenance or insertion practices. CONCLUSION: There is substantial variation in reported CA-BSI surveillance practices among PICUs, and more aggressive surveillance correlates to higher CA-BSI rates, which has important implications in pay-for-performance and benchmarking applications. There is a compelling opportunity to improve standardized CA-BSI surveillance to enhance the validity of this metric for interinstitutional comparisons. Health care professionals' attitudes and beliefs about CA-BSI being driven by patient risk factors would benefit from recalibration that emphasized more important drivers-such as the quality of central line insertion and maintenance.

Rapid identification of infants for antiretroviral therapy in a resource poor setting: the Kenya experience.

In Kenya, HIV diagnosis is not routinely carried out in infants, and yet rapid diagnosis could improve access to lifesaving interventions. A cheap and readily accessible service can resolve this problem, if feasible. In this pilot study the feasibility and costs of provision of an infant HIV diagnosis service in Kenya are evaluated. Dried blood spots (DBS) were collected from infants exposed to HIV, sent to a central testing laboratory and tested using the Roche Amplicor v. 1.5 DNA PCR kit. The results were then dispatched to health facilities within a week. A total of 15.4% of the samples tested HIV+ despite the widespread access to prevention of mother to child transmission (PMTCT) programs in Kenya. The cost per test at 21.50 USD is prohibitive and will limit access to diagnosis. It remains to be seen whether the increase in testing will immediately lead to an increase in access to antiretroviral therapy (ART) services for infants.