Pathogens that Cause Illness Clinically Indistinguishable from Lassa Fever, Nigeria, 2018.

During the 2018 Lassa fever outbreak in Nigeria, samples from patients with suspected Lassa fever but negative Lassa virus PCR results were processed through custom gene expression array cards and metagenomic sequencing. Results demonstrated no single etiology, but bacterial and viral pathogens (including mixed co-infections) were detected.

Histopathology Is Key to Interpreting Multiplex Molecular Test Results From Postmortem Minimally Invasive Tissue Samples.

BACKGROUND: Minimally invasive tissue sampling (MITS) is an alternative to complete autopsy for determining causes of death. Multiplex molecular testing performed on MITS specimens poses challenges of interpretation, due to high sensitivity and indiscriminate detection of pathogenic, commensal, or contaminating microorganisms. METHODS: MITS was performed on 20 deceased children with respiratory illness, at 10 timepoints up to 88 hours postmortem. Samples were evaluated by multiplex molecular testing on fresh tissues by TaqMan® Array Card (TAC) and by histopathology, special stains, immunohistochemistry (IHC), and molecular testing (PCR) on formalin-fixed, paraffin-embedded (FFPE) tissues. Results were correlated to determine overall pathologic and etiologic diagnoses and to guide interpretation of TAC results. RESULTS: MITS specimens collected up to 3 days postmortem were adequate for histopathologic evaluation and testing. Seven different etiologic agents were detected by TAC in 10 cases. Three cases had etiologic agents detected by FFPE or other methods and not TAC; 2 were agents not present on TAC, and 2 were streptococci that may have been species other than those present on TAC. Result agreement was 43% for TAC and IHC or PCR, and 69% for IHC and PCR. Extraneous TAC results were common, especially when aspiration was present. CONCLUSIONS: TAC can be performed on MITS up to 3 days after death with refrigeration and provides a sensitive method for detection of pathogens but requires careful interpretation in the context of clinicoepidemiologic and histopathologic findings. Interpretation of all diagnostic tests in aggregate to establish overall case diagnoses maximizes the utility of TAC in MITS.

Evaluation of TaqMan Array card (TAC) for the detection of 28 respiratory pathogens.

BACKGROUND: Respiratory infections are a serious threat to human health. So, rapid detection of all respiratory pathogens can facilitate prompt treatment and prevent the deterioration of respiratory disease. Previously published primers and probes of the TaqMan array card (TAC) for respiratory pathogens are not sensitive to Chinese clinical specimens. This study aimed to develop and improve the TAC assay to detect 28 respiratory viral and bacterial pathogens in a Chinese population. METHODS: To improve the sensitivity, we redesigned the primers and probes, and labeled the probes with minor groove binders. The amplification efficiency, sensitivity, and specificity of the primers and probes were determined using target-gene containing standard plasmids. The detection performance of the TAC was evaluated on 754 clinical specimens and the results were compared with those from conventional methods. RESULTS: The performance of the TAC assay was evaluated using 754 clinical throat swab samples and the results were compared with those from gold-standard methods. The sensitivity and specificity were 95.4 and 96.6%, respectively. The lowest detection limit of the TAC was 10 to 100 copies/µL. CONCLUSIONS: TAC is an efficient, accurate, and high-throughput approach to detecting multiple respiratory pathogens simultaneously and is a promising tool for the identification of pathogen outbreaks.

Development and Implementation of Multiplex TaqMan Array Cards for Specimen Testing at Child Health and Mortality Prevention Surveillance Site Laboratories.

Child Health and Mortality Prevention Surveillance (CHAMPS) laboratories are employing a variety of laboratory methods to identify infectious agents contributing to deaths of children <5 years old and stillbirths in sub-Saharan Africa and South Asia. In support of this long-term objective, our team developed TaqMan Array Cards (TACs) for testing postmortem specimens (blood, cerebrospinal fluid, lung tissue, respiratory tract swabs, and rectal swabs) for >100 real-time polymerase chain reaction (PCR) targets in total (30-45 per card depending on configuration). Multipathogen panels were configured by syndrome and customized to include pathogens of significance in young children within the regions where CHAMPS is conducted, including bacteria (57 targets covering 30 genera), viruses (48 targets covering 40 viruses), parasites (8 targets covering 8 organisms), and fungi (3 targets covering 3 organisms). The development and application of multiplex real-time PCR reactions to the TAC microfluidic platform increased the number of targets in each panel while maintaining assay efficiency and replicates for heightened sensitivity. These advances represent a substantial improvement in the utility of this technology for infectious disease diagnostics and surveillance. We optimized all aspects of the CHAMPS molecular laboratory testing workflow including nucleic acid extraction, quality assurance, and data management to ensure comprehensive molecular testing of specimens and high-quality data. Here we describe the development and implementation of multiplex TACs and associated laboratory protocols for specimen processing, testing, and data management at CHAMPS site laboratories.

Development of a TaqMan Array card to target 21 purulent meningitis-related pathogens.

BACKGROUND: Purulent meningitis (PM) is a serious life-threatening infection of the central nervous system (CNS) by bacteria or fungi and associated with high mortality and high incidence of CNS sequelae in children. However, the conventional cerebrospinal fluid (CSF) culture method is time-consuming and has a low sensitivity. METHODS: Our study developed a real-time PCR-based purulent meningitis-TaqMan array card (PM-TAC) that targeted 21 PM-related pathogens and could produce results within 3 h. Primers and probes were adapted from published sources possibly. The performance of them were evaluated and optimized and then they were spotted on TAC. RESULTS: The PM-TAC showed a sensitivity and specificity of 95 and 96%, respectively. For all of the 21 targeted pathogens, the PM-TAC assay had a LOD ranging from 5 copies/reaction to 100 copies/reaction, an intra-assay variation of 0.07-4.45%, and an inter-assay variation of 0.11-6.81%. Of the 15 CSF samples collected from patients with PM after empiric antibiotic therapies, the positive rate was 53.3% (8/15) for our PM-TAC assay but was only 13.3% (2/15) for the CSF culture method. Of the 17 CSF samples showing negative CSF culture, the PM-TAC assay identified a case of Neisseria meningitidis infection. Furthermore, all of the 10 CSF samples from patients without CNS infection showed negative for the PM-TAC assay. CONCLUSIONS: Our PM-TAC assay also demonstrated that the pathogen loads in the CSF samples correlated with the severity of PM. Thus, the PM-TAC may be helpful to improve the prognosis of PM and clinical outcomes from antibiotic therapies.

Building laboratory capacity to detect and characterize pathogens of public and global health security concern in Kenya.

Since 1979, multiple CDC Kenya programs have supported the development of diagnostic expertise and laboratory capacity in Kenya. In 2004, CDC's Global Disease Detection (GDD) program within the Division of Global Health Protection in Kenya (DGHP-Kenya) initiated close collaboration with Kenya Medical Research Institute (KEMRI) and developed a laboratory partnership called the Diagnostic and Laboratory Systems Program (DLSP). DLSP built onto previous efforts by malaria, human immunodeficiency virus (HIV) and tuberculosis (TB) programs and supported the expansion of the diagnostic expertise and capacity in KEMRI and the Ministry of Health. First, DLSP developed laboratory capacity for surveillance of diarrheal, respiratory, zoonotic and febrile illnesses to understand the etiology burden of these common illnesses and support evidenced-based decisions on vaccine introductions and recommendations in Kenya. Second, we have evaluated and implemented new diagnostic technologies such as TaqMan Array Cards (TAC) to detect emerging or reemerging pathogens and have recently added a next generation sequencer (NGS). Third, DLSP provided rapid laboratory diagnostic support for outbreak investigation to Kenya and regional countries. Fourth, DLSP has been assisting the Kenya National Public Health laboratory-National Influenza Center and microbiology reference laboratory to obtain World Health Organization (WHO) certification and ISO15189 accreditation respectively. Fifth, we have supported biosafety and biosecurity curriculum development to help Kenyan laboratories safely and appropriately manage infectious pathogens. These achievements, highlight how in collaboration with existing CDC programs working on HIV, tuberculosis and malaria, the Global Health Security Agenda can have significantly improve public health in Kenya and the region. Moreover, Kenya provides an example as to how laboratory science can help countries detect and control of infectious disease outbreaks and other public health threats more rapidly, thus enhancing global health security.

Detection of Dengue Virus among Children with Suspected Malaria, Accra, Ghana.

We report new molecular evidence of locally acquired dengue virus infections in Ghana. We detected dengue viral RNA among children with suspected malaria by using a multipathogen real-time PCR. Subsequent sequence analysis revealed a close relationship with dengue virus serotype 2, which was implicated in a 2016 outbreak in Burkina Faso.

Improved Detection of Respiratory Pathogens by Use of High-Quality Sputum with TaqMan Array Card Technology.

New diagnostic platforms often use nasopharyngeal or oropharyngeal (NP/OP) swabs for pathogen detection for patients hospitalized with community-acquired pneumonia (CAP). We applied multipathogen testing to high-quality sputum specimens to determine if more pathogens can be identified relative to NP/OP swabs. Children (<18 years old) and adults hospitalized with CAP were enrolled over 2.5 years through the Etiology of Pneumonia in the Community (EPIC) study. NP/OP specimens with matching high-quality sputum (defined as ≤10 epithelial cells/low-power field [lpf] and ≥25 white blood cells/lpf or a quality score [q-score] definition of 2+) were tested by TaqMan array card (TAC), a multipathogen real-time PCR detection platform. Among 236 patients with matched specimens, a higher proportion of sputum specimens had ≥1 pathogen detected compared with NP/OP specimens in children (93% versus 68%; P < 0.0001) and adults (88% versus 61%; P < 0.0001); for each pathogen targeted, crossing threshold (CT) values were earlier in sputum. Both bacterial (361 versus 294) and viral detections (245 versus 140) were more common in sputum versus NP/OP specimens, respectively, in both children and adults. When available, high-quality sputum may be useful for testing in hospitalized CAP patients.

Spatial-Temporal Patterns in the Enteric Pathogen Contamination of Soil in the Public Environments of Low- and Middle-Income Neighborhoods in Nairobi, Kenya.

Public spaces in countries with limited societal development can be contaminated with feces containing pathogenic microbes from animals and people. Data on contamination levels, spatial distribution, and the diversity of enteric pathogens in the public settings of low- and middle-income neighborhoods are crucial for devising strategies that minimize the enteric infection burden. The objective of this study was to compare spatial-temporal differences in the detection rate and diversity of enteric pathogens in the public spaces of low- and middle-income neighborhoods of Nairobi, Kenya. TaqMan array card (TAC) molecular assays were employed to analyze soil samples for 19 enteropathogens, along with a selective bacterial culture for pathogenic Enterobacteriaceae. An observational assessment was conducted during every site visit to document the hygienic infrastructure and sanitation conditions at the sites. We detected at least one pathogen in 79% (127/160) and ≥2 pathogens in 67.5% (108/160) of the soil samples tested. The four most frequently detected pathogens were EAEC (67.5%), ETEC (59%), EPEC (57.5%), and STEC (31%). The detection rate (91% vs. 66%) and mean number of enteric pathogens (5 vs. 4.7) were higher in low-income Kibera than in middle-income Jericho. The more extensive spatial distribution of pathogens in Kibera resulted in increases in the detection of different enteric pathogens from within-site (area < 50 m2) and across-site (across-neighborhood) movements compared to Jericho. The pathogen detection rates fluctuated seasonally in Jericho but remained at sustained high levels in Kibera. While better neighborhood conditions were linked with lower pathogen detection rates, pathogenic E. coli remained prevalent in the public environment across both neighborhoods. Future studies should focus on identifying how the sources of pathogen contamination are modified by improved environmental sanitation and hygiene and the role of these contaminated public environments in enteric infections in children.

Differences in epidemiology of enteropathogens in children pre- and post-rotavirus vaccine introduction in Kilifi, coastal Kenya.

BACKGROUND: Kenya introduced Rotarix® (GlaxoSmithKline Biologicals, Rixensart, Belgium) vaccination into its national immunization programme beginning July 2014. The impact of this vaccination program on the local epidemiology of various known enteropathogens is not fully understood. METHODS: We used a custom TaqMan Array Card (TAC) to screen for 28 different enteropathogens in 718 stools from children aged less than 13 years admitted to Kilifi County Hospital, coastal Kenya, following presentation with diarrhea in 2013 (before vaccine introduction) and in 2016-2018 (after vaccine introduction). Pathogen positivity rate differences between pre- and post-Rotarix® vaccination introduction were examined using both univariate and multivariable logistic regression models. RESULTS: In 665 specimens (92.6%), one or more enteropathogen was detected, while in 323 specimens (48.6%) three or more enteropathogens were detected. The top six detected enteropathogens were: enteroaggregative Escherichia coli (EAggEC; 42.1%), enteropathogenic Escherichia coli (EPEC; 30.2%), enterovirus (26.9%), rotavirus group A (RVA; 24.8%), parechovirus (16.6%) and norovirus GI/GII (14.4%). Post-rotavirus vaccine introduction, there was a significant increase in the proportion of samples testing positive for EAggEC (35.7% vs. 45.3%, p = 0.014), cytomegalovirus (4.2% vs. 9.9%, p = 0.008), Vibrio cholerae (0.0% vs. 2.3%, p = 0.019), Strongyloides species (0.8% vs. 3.6%, p = 0.048) and Dientamoeba fragilis (2.1% vs. 7.8%, p = 0.004). Although not reaching statistical significance, the positivity rate of adenovirus 40/41 (5.8% vs. 7.3%, p = 0.444), norovirus GI/GII (11.2% vs. 15.9%, p = 0.089), Shigella species (8.7% vs. 13.0%, p = 0.092) and Cryptosporidium spp. (11.6% vs. 14.7%, p = 0.261) appeared to increase post-vaccine introduction. Conversely, the positivity rate of sapovirus decreased significantly post-vaccine introduction (7.8% vs. 4.0%, p = 0.030) while that of RVA appeared not to change (27.4% vs. 23.5%, p = 0.253). More enteropathogen coinfections were detected per child post-vaccine introduction compared to before (mean: 2.7 vs. 2.3; p = 0.0025). CONCLUSIONS: In this rural Coastal Kenya setting, childhood enteropathogen infection burden was high both pre- and post-rotavirus vaccination introduction. Children who had diarrheal admissions post-vaccination showed an increase in coinfections and changes in specific enteropathogen positivity rates. This study highlights the utility of multipathogen detection platforms such as TAC in understanding etiology of childhood acute gastroenteritis in resource-limited regions.

Fast screening of enteropathogens in marine water samples.

This study aimed to fast screen the microbiological contamination of recreational waters using a TaqMan Array Card (TAC), a multiplexed platform designed for the simultaneous detection of 35 enteropathogens. Surface and deep marine water samples were concentrated by skimmed milk flocculation and processed for nucleic acid extraction protocol using QIAamp Fast DNA Stool Mini Kit. Twelve microorganisms and parasites, including bacteria (n = 6), protozoa (4), and viruses (2), were detected in 85.7% (24/28) of samples. Campylobacter (82.1%), Cryptosporidium (39.3%), and adenovirus (14.3%) were the most detected pathogens. Neither fungi nor helminths were detected. A spatial pollution profile of microbiological contamination was observed in the area. Methodologies for simultaneous detection of multiple pathogens, such as TAC, can assist decision-makers by providing a quick assessment of the microbiological water quality in areas used for recreational purposes, which in many cases are in accordance with the bacteriological indicators.

TaqMan Array Card testing of participant-collected stool smears to determine the pathogen-specific epidemiology of travellers' diarrhoea†.

BACKGROUND: We assessed the compliance with self-collection of stool smears on Whatman® FTA® Elute Card (FTA Card) and detection of travellers' diarrhoea (TD)-associated pathogens by using a quantitative Polymerase Chain Reaction (PCR) assay [customized TaqMan® array card (TAC)] in a prospective, observational cohort of travellers. METHODS: Enrolled travellers documented symptoms on a travel diary and collected an FTA Card during a diarrhoeal episode, or at the end of travel if they remained asymptomatic. TAC testing was performed on FTA Cards from TD cases and 1:1 matched asymptomatic controls and 1:1 matched loose stool cases that did not meet TD criteria. Odds ratios were used to determine the association between detected pathogens and TD. RESULTS: Of 2456 travellers, 484 (19.7%) completed an illness diary and met TD criteria, and 257 (53.1%) collected an FTA Card during the TD episode. FTA Cards were stored for a median of 2 years at room temperature (IQR: 1-4 years) before extraction and testing. The overall TAC detection rate in TD cases was 58.8% (95% CI: 52.5-64.8). Enterotoxigenic Escherichia coli was the most common pathogen in TD cases (26.8%), and 3.5% of samples were positive for norovirus. The odds of detecting TD-associated pathogens in 231 matched cases and asymptomatic controls were 5.4 (95% CI: 3.6-8.1) and 2.0 (95% CI: 1.1-3.7) in 121 matched TD and loose stool cases (P < 0.05). Enteroaggregative E. coli was the most common pathogen detected in asymptomatic controls and loose stool cases. Detection of diarrhoeagenic E. coli, Shigella/enteroinvasive E. coli and Campylobacter spp. was significantly associated with TD. CONCLUSION: FTA Cards are a useful adjunct to traditional stool collection methods for evaluating the pathogen-specific epidemiology of TD in austere environments. Qualitative detection of pathogens was associated with TD. Measures to improve compliance and quality of FTA Card collection with decreased storage duration may further optimize detection.

Impact of enteropathogens on faltering growth in a resource-limited setting.

Introduction: Environmental enteropathy is an important contributor to childhood malnutrition in the developing world. Chronic exposure to fecal pathogens leads to alteration in intestinal structure and function, resulting in impaired gut immune function, malabsorption, and growth faltering leading to environmental enteropathy. Methods: A community-based intervention study was carried out on children till 24 months of age in Matiari district, Pakistan. Blood and fecal specimens were collected from the enrolled children aged 3-6 and 9 months. A real-time PCR-based TaqMan array card (TAC) was used to detect enteropathogens. Results: Giardia, Campylobacter spp., enteroaggregative Escherichia coli (EAEC), Enteropathogenic Escherichia coli (EPEC), Enterotoxigenic Escherichia coli (ETEC), and Cryptosporidium spp. were the most prevailing enteropathogens in terms of overall positivity at both time points. Detection of protozoa at enrollment and 9 months was negatively correlated with rate of change in height-for-age Z (ΔHAZ) scores during the first and second years of life. A positive association was found between Giardia, fecal lipocalin (LCN), and alpha 1-Acid Glycoprotein (AGP), while Campylobacter spp. showed positive associations with neopterin (NEO) and myeloperoxidase (MPO). Conclusion: Protozoal colonization is associated with a decline in linear growth velocity during the first 2 years of life in children living in Environmental enteric dysfunction (EED) endemic settings. Mechanistic studies exploring the role of cumulative microbial colonization, their adaptations to undernutrition, and their influence on gut homeostasis are required to understand symptomatic enteropathogen-induced growth faltering.

Pathogens detected using a syndromic molecular diagnostic platform in patients hospitalized with severe respiratory illness in South Africa in 2017.

OBJECTIVES: We describe the use of a multipathogen platform, TaqMan array card (TAC) real-time polymerase chain reaction, for the detection of pathogens in patients hospitalized with severe respiratory illness (SRI). METHODS: Prospective hospital-based syndromic surveillance for acute and chronic SRI was carried out at two sentinel sites in South Africa between January and December 2017. We tested respiratory specimens for 21 respiratory pathogens and blood samples for nine bacteria using TAC. Pathogen detection was compared by age group and HIV status using the chi-square test. RESULTS: During 2017, 956 patients of all ages were enrolled in the SRI surveillance, and of these, 637 (67%) patients were included in this study (637 blood, 487 naso- and oro-pharyngeal swabs, and 411 sputum specimens tested). At least one pathogen was detected in 83% (527/637) of patients. Common pathogens detected included Haemophilus influenzae (225/637; 35%), Streptococcus pneumoniae (224/637; 35%), rhinovirus (144/637; 23%), Staphylococcus aureus (129/637; 20%), Klebseilla pneumoniae (85/637; 13%), Mycobacterium tuberculosis (75/637; 12%), and respiratory syncytial virus (57/637; 9%). Multiple pathogens (≥2) were codetected in 57% (364/637) of patients. CONCLUSION: Although the use of a multi-pathogen platform improved pathogen yield, pathogen co-detections were common and would need clinical assessment for usefulness in individual-level treatment and management decisions.

Microbiological assessment of an urban lagoon system in the coastal zone of Rio de Janeiro, Brazil.

This study aims to assess microbiological contamination using a molecular tool for detection of multiple enteropathogens in a coastal ecosystem area in Rio de Janeiro, Brazil. Ten litres of superficial water samples were obtained during the spring ebb tide from sampling sites along the Jacarepaguá watershed. Samples were concentrated using skimmed milk flocculation method for TaqMan array card (TAC), designed to identify 35 enteric pathogens simultaneously, and single TaqMan qPCR analysis for detecting human adenovirus (HAdV) and JC human polyomavirus (JCPyV) as faecal indicator viruses (FIV). TAC results identified 17 enteric pathogens including 4/5 viral species investigated, 8/15 bacteria, 4/6 protozoa and 1/7 helminths. Escherichia coli concentration was also measured as faecal indicator bacteria (FIB) using Colilert Quanti-Tray System with positivity in all samples studied. HAdV and JCPyV qPCR were detected in 8 and 4 samples, respectively, with concentration ranging from 8 × 102 to 2 × 106 genome copies/L. Partial nucleotide sequencing demonstrated the occurrence of species HAdV A, C, D, and F, present in faeces of individuals with enteric and non-enteric infections, and JCPyV type 3 (Af2), prevalent in a high genetically mixed population like the Brazilian. The diversity of enteropathogens detected by TAC emphasizes the utility of this methodology for quick assessment of microbiological contamination of the aquatic ecosystems, speeding up mitigation actions where the risk of the exposed population is detected, as well as pointing out the infrastructure gaps in areas where accelerated urban growth is observed.

Enteric Pathogen Diversity in Infant Foods in Low-Income Neighborhoods of Kisumu, Kenya.

Pediatric diarrheal disease remains the second most common cause of preventable illness and death among children under the age of five, especially in low and middle-income countries (LMICs). However, there is limited information regarding the role of food in pathogen transmission in LMICs. For this study, we examined the frequency of enteric pathogen occurrence and co-occurrence in 127 infant weaning foods in Kisumu, Kenya, using a multi-pathogen PCR diagnostic tool, and assessed household food hygiene risk factors for contamination. Bacterial, viral, and protozoan enteric pathogen DNA and RNA were detected in 62% of the infant weaning food samples collected, with 37% of foods containing more than one pathogen type. Multivariable generalized linear mixed model analysis indicated type of infant food best explained the presence and diversity of enteric pathogens in infant food, while most household food hygiene risk factors considered in this study were not significantly associated with pathogen contamination. Specifically, cow's milk was significantly more likely to contain a pathogen (adjusted risk ratio = 14.4; 95% confidence interval (CI) 1.78⁻116.1) and more likely to have higher number of enteric pathogen species (adjusted risk ratio = 2.35; 95% CI 1.67⁻3.29) than porridge. Our study demonstrates that infants in this low-income urban setting are frequently exposed to diarrhoeagenic pathogens in food and suggests that interventions are needed to prevent foodborne transmission of pathogens to infants.

Comparison of Minimally Invasive Tissue Sampling With Conventional Autopsy to Detect Pulmonary Pathology Among Respiratory Deaths in a Resource-Limited Setting.

OBJECTIVES: We compared minimally invasive tissue sampling (MITS) with conventional autopsy (CA) in detection of respiratory pathology/pathogens among Kenyan children younger than 5 years who were hospitalized with respiratory disease and died during hospitalization. METHODS: Pulmonary MITS guided by anatomic landmarks was followed by CA. Lung tissues were triaged for histology and molecular testing using TaqMan Array Cards (TACs). MITS and CA results were compared for adequacy and concordance. RESULTS: Adequate pulmonary tissue was obtained by MITS from 54 (84%) of 64 respiratory deaths. Comparing MITS to CA, full histologic diagnostic concordance was present in 23 (36%) cases and partial concordance in 19 (30%), an overall 66% concordance rate. Pathogen detection using TACs had full concordance in 27 (42%) and partial concordance in 24 (38%) cases investigated, an overall 80% concordance rate. CONCLUSIONS: MITS is a viable alternative to CA in respiratory deaths in resource-limited settings, especially if combined with ancillary tests to optimize diagnostic accuracy.

Performance evaluation of direct fluorescent antibody, Focus Diagnostics Simplexa™ Flu A/B & RSV and multi-parameter customized respiratory Taqman® array card in immunocompromised patients.

BACKGROUND: Molecular assays for diagnosis of Flu A, Flu B, and RSV with short turn-around-time (TAT) are of considerable clinical importance. In addition, rapid and accurate diagnosis of a large panel of viral and atypical pathogens can be crucial in immunocompromised patients. OBJECTIVES: First, to evaluate the performance of the Simplexa™ Direct assay system in comparison with direct fluorescent antibody (DFA) and customized Taqman® Array Card (TAC) testing for RSV, Flu A, and Flu B in immunocompromised patients. Second, to evaluate different algorithms for the detection of respiratory pathogens in terms of cost, turn-around-time (TAT) and diagnostic yield. STUDY DESIGN: We collected 125 nasopharyngeal swabs (NTS) and 25 BAL samples from symptomatic immunocompromised patients. Samples for which Simplexa™ and TAC results were discordant underwent verification testing. The TAC assay is based on singleplex RT-PCR, targeting 24 viruses, 8 bacteria and 2 fungi simultaneously. RESULTS: The overall sensitivity was significantly lower for DFA testing than for the two molecular methods (p<0.05). Performance characteristics of Simplexa™ testing were not significantly different compared to TAC testing (p>0.1). For BAL samples only, the sensitivity and specificity of the Simplexa™ assay was 100%. In total, 6.7, 16 and 18% of samples were positive for Flu A, Flu B or RSV by DFA, Simplexa™ and TAC testing, respectively. When considering not only these pathogens but also all results for TAC, the method identified 93 samples with one or more respiratory pathogens (62%). A co-infection rate of 15.3% was found by TAC. The estimated costs and TAT were 8.2€ and 2h for DFA, 31.8€ and 1.5h for Simplexa™ and 55€ and 3h for TAC testing. CONCLUSIONS: Performing the Simplexa™ test 24h a day/7 days a week instead of DFA would considerably improve the overall sensitivity and time-to-result, albeit at a higher cost generated in the laboratory. Performing the TAC would increase the diagnostic yield and detection of co-infections significantly.