Direct Fluorescent Antibody Staining Research Articles

Diagnosis of Legionnaires' Disease Assisted by Next-Generation Sequencing in a Patient with COVID-19.

Coinfection in COVID-19 patients is associated with worsening outcome. Among patients with COVID-19, Legionella pneumophila, a common cause of pneumonia, has been reported as a co-occurring respiratory infection. A nonspecific clinical presentation, however, makes an early diagnosis difficult. Bronchoalveolar lavage fluid was collected from a patient suffering from COVID-19 and presenting with pneumonia and sent for metagenomic analysis. Differential abundance analysis was carried out by comparing each taxon reads per million between the bronchoalveolar lavage fluid sample and the negative control. Two replicates of metagenomic sequencing were conducted on bronchoalveolar lavage fluid samples. Within each replicated sequencing, one negative control was sequenced for comparison of taxon abundance in the BALF sample. In both replicates, Legionella pneumophila was the only taxon with significantly higher abundance when compared with the negative control. PCR of the bronchoalveolar further confirmed the presence of L. pneumophila. Several studies have estimated that the incidence of Legionnaires' disease co-infection in patients with COVID-19 infection is approximately 0% to 1.5%. There are some common characteristics of COVID-19 and co-infection with Legionnaires' disease, making it difficult to diagnose bacterial infection early. The diagnosis of these cases is important due to the different treatments used. Current diagnostic tests for Legionnaires' disease include conventional culture, urinary antigen for L. pneumophila serogroup 1, polymerase chain reaction, direct fluorescent antibody stain, and paired serology. The current study demonstrated that metagenomics is a promising approach that facilitated the diagnosis of Legionnaires' disease.

Rapidly Progressive Cognitive Decline and Varicella Zoster Blisters Associated With Teprotumumab in 2 Patients With Graves’ Orbitopathy

Introduction: Teprotumumab, an IGF-1R antagonist, was approved for treatment of Graves’ orbitopathy (GO) in 2020. Common side effects include nausea, diarrhea, muscle spasms, hearing impairment, dysgeusia, headache, dry skin, infusion reactions, and hyperglycemia. We report here 2 cases of unexpected adverse effects. Patient #1 is a 76-year-old man with Graves’ disease diagnosed in 2018, treated with methimazole 2.5mg every other day. His GO manifestations included diplopia, proptosis, and exposure keratopathy. Past history was significant for hypertension, hyperlipidemia, and prostate hyperplasia. He lived independently and walked several miles daily. Teprotumumab was initiated. Between the 4th and 5th doses, his family noted 6-weeks of rapidly progressive cognitive decline characterized by behavioral changes, confabulation, memory deficit, delirium/delusions/mania. He was admitted to neurology service and subsequent doses held. Lumbar puncture was unremarkable. TFT was normal before, during and after stopping teprotumumab. Thyroid antibodies were notable for elevated TSI but normal thyrotropin receptor Ab and TPO/TgAb. Other labs: IL2, IL6, ESR, CRP, heavy metals, IgG, IgM, SARS-CoV-2 IgM/IgGs, vitamins B1, B6, and B12 were all normal. His most recent repeat TPO Ab was 14 IU/mL (increased from <6), TgAb 18.4 IU/mL (increased from <1.0). EEG showed no epileptiform activity. Head MRI/MRA showed cerebral amyloid angiopathy without stenosis or aneurysms in the intracranial arterial vasculature. Symptoms progressed with IV glucocorticoids and IV Ig therapy. He subsequently underwent plasmapheresis with resolution of symptoms. Patient #2 is a 48-year-old man with Graves’ disease and GO diagnosed in 2018, initially treated with methimazole and subsequently underwent total thyroidectomy in 2019. Patient is euthyroid on levothyroxine. Ophthalmic signs and symptoms included significant proptosis and retrobulbar pain. He was started on teprotumumab infusions. After his 4th dose, he noted painful vesicles in a dermatomal distribution on his right posterior thigh. Direct fluorescent antibody stain was positive for varicella zoster, and rashes resolved with valacyclovir therapy. Discussion: Paraneoplastic/autoimmune encephalitis syndromes can be associated with antibodies to neuronal cell surfaces/synaptic proteins and may occur in the presence or absence of cancer. The incidence of varicella-zoster virus infection is associated with the immune status of the patient, disease-related immunocompromised, or iatrogenic immunosuppression, with age being the major risk factor for 90% of adult cases. This could be incidental, or related to other disorders, or perhaps teprotumumab may have activated auto-immunity. The exact mechanisms for rapidly progressive cognitive decline and shingles remains unclear.

Effects of mannan-oligosaccharide and Bacillus subtilis supplementation to preweaning Holstein dairy heifers on body weight gain, diarrhea, and shedding of fecal pathogens

The objective of this clinical trial was to evaluate the effectiveness of probiotic, prebiotic, and synbiotic supplementation on average daily weight gain (ADG), duration of diarrhea, age at incidence of diarrhea, fecal shedding of Cryptosporidium oocysts, enteric pathogens, and the odds of pneumonia in preweaning dairy heifer calves on a commercial dairy. Feeding prebiotics and probiotics may improve health and production of calves. Hence, healthy Holstein heifer calves (n = 1,801) from a large California dairy were enrolled at 4 to 12 h of age and remained in this study until weaning at 60 d of age. Calves were block randomized to 1 of 4 treatments: (1) control, (2) yeast culture enriched with mannan-oligosaccharide (prebiotic), (3) Bacillus subtilis (probiotic), and (4) combination of both products (synbiotic), which were fed in milk twice daily from enrollment until weaning. Serum total protein at enrollment and body weight at 7, 42, and 56 d of age were measured. Fecal consistency was assessed daily for the entire preweaning period. A subgroup of 200 calves had fecal samples collected at 7, 14, 21, and 42 d for microbial culture and enumeration of Cryptosporidium oocysts by direct fluorescent antibody staining. Synbiotic-treated calves had 19 g increased ADG compared with control calves for overall ADG, from 7 to 56 d. From 42 to 56 d, prebiotic-treated calves had 85 g greater ADG and synbiotic-treated calves had 78 g greater ADG than control calves. There was no difference in duration of the first diarrhea episode, hazard of diarrhea, or odds of pneumonia per calf with treatment. Probiotic-treated calves had 100 times lower fecal shedding of Cryptosporidium oocysts at 14 d and prebiotic-treated calves had fewer Escherichia coli and pathogenic E. coli at 42 d compared with control calves. Although there were no effects on duration of diarrhea or pneumonia incidence, greater ADG in the late preweaning period may reflect treatment effects on enteric pathogens during the rearing process. The decreased shedding of Cryptosporidium should reduce infectious pressure, environmental contamination, and public health risks from Cryptosporidium. Our findings suggest ADG and potential health benefits for calves fed prebiotics, probiotics, and synbiotics and can help the dairy industry make informed decisions on the use of these products in dairy production.

Laboratory Diagnostic Tools for Syphilis: Current Status and Future Prospects.

With the increasing number of patients infected with syphilis in the past 20 years, early diagnosis and early treatment are essential to decline syphilis prevalence. Owing to its diverse manifestations, which may occur in other infections, the disease often makes clinicians confused. Therefore, a sensitive method for detecting T. pallidum is fundamental for the prompt diagnosis of syphilis. Morphological observation, immunohistochemical assay, rabbit infectivity test, serologic tests, and nucleic acid amplification assays have been applied to the diagnosis of syphilis. Morphological observation, including dark-field microscopy, silver-staining, and direct fluorescent antibody staining for T. pallidum, can be used as a direct detection method for chancre specimens in primary syphilis. Immunohistochemistry is a highly sensitive and specific assay, especially in the lesion biopsies from secondary syphilis. Rabbit infectivity test is considered as a sensitive and reliable method for detecting T. pallidum in clinical samples and used as a historical standard for the diagnosis of syphilis. Serologic tests for syphilis are widely adopted using non-treponemal or treponemal tests by either the traditional or reverse algorithm and remain the gold standard in the diagnosis of syphilis patients. In addition, nucleic acid amplification assay is capable of detecting T. pallidum DNA in the samples from patients with syphilis. Notably, PCR is probably a promising method but remains to be further improved. All of the methods mentioned above play important roles in various stages of syphilis. This review aims to provide a summary of the performance characteristics of detection methods for syphilis.

Central Nervous System Disease and Adenoviruses

Research Article| March 01 2019 Central Nervous System Disease and Adenoviruses AAP Grand Rounds (2019) 41 (3): 35. https://doi.org/10.1542/gr.41-3-35 Views Icon Views Article contents Figures & tables Video Audio Supplementary Data Peer Review Share Icon Share Facebook Twitter LinkedIn MailTo Tools Icon Tools Get Permissions Cite Icon Cite Search Site Citation Central Nervous System Disease and Adenoviruses. AAP Grand Rounds March 2019; 41 (3): 35. https://doi.org/10.1542/gr.41-3-35 Download citation file: Ris (Zotero) Reference Manager EasyBib Bookends Mendeley Papers EndNote RefWorks BibTex toolbar search toolbar search search input Search input auto suggest filter your search All PublicationsAll JournalsAAP Grand RoundsPediatricsHospital PediatricsPediatrics In ReviewNeoReviewsAAP NewsAll AAP Sites Search Advanced Search Topics: adenoviruses, central nervous system dysfunction, adenovirus infections Source: Schwartz KL, Richardson SE, MacGregor D, et al. Adenovirus-associated central nervous system disease in children. J Pediatr. 2018 6 November [published online ahead of print]; doi: https://doi.org/10.1016/j.jpeds.2018.09.036Google Scholar Investigators from The Hospital for Sick Children (SickKids) in Toronto conducted a retrospective observational study to characterize adenovirus-associated central nervous system (CNS) disease in children. Three data sources were used: the SickKids microbiology and encephalitis databases (which include children who had been admitted to SickKids during 2000–2016 and 1996–2016, respectively) and a literature search. Children in the SickKids databases were eligible for the study if they were previously healthy; were 1 month to 18 years old; had neurologic symptoms; did not have a preexisting neurologic disorder; and had adenovirus detected by direct fluorescent antibody staining, culture, or polymerase chain reaction (PCR). Child cases identified from the literature search were eligible if they had a neurologic diagnosis other than febrile seizures and had microbiologic or serologic evidence of acute adenovirus infection. Clinical and demographic characteristics of identified children were abstracted from each data source. Using SickKids microbiology data, investigators determined the frequency and spectrum of neurologic complications of adenovirus disease. Using SickKids encephalitis data, investigators determined the frequency of adenovirus among children with encephalitis. Cases identified by literature search were then pooled with children identified in the SickKids databases (excluding those with febrile seizures) to characterize the adenovirus-associated CNS disease spectrum further. Multivariable logistic regression was used to identify risk factors associated with death or neurologic impairment. Of the 977 cases of adenovirus infection in the SickKids microbiology database, 32 (3.3%) had neurologic symptoms. Of these, 26 had febrile seizures, 4 had encephalitis, 1 had acute demyelinating encephalomyelitis, and 1 had aseptic meningitis. Of the 431 children in the SickKids encephalitis database, 8 (1.9%) had adenovirus (including the 4 with encephalitis identified in the SickKids microbiology database). There were 16 studies identified by the literature search involving 38 children, resulting in a pooled total of 48 cases. Of these, the median age was 2 years, and 40% were female. The most common prodromal symptoms were upper respiratory illness (48%), vomiting (22%), and diarrhea (19%). Hepatitis developed during hospitalization in 40%. There were 10 (21%) deaths, and 8 (17%) had permanent neurologic impairment. Children who developed seizures during their illness course had greater odds of death or permanent neurologic impairment (adjusted OR, 7.3; 95% CI, 1.69–31.45). The investigators conclude that adenovirus is a rare cause of CNS disease in children but can be fatal. Dr Brady has disclosed no financial relationship relevant to this commentary. This commentary does not contain a discussion of an unapproved/investigative use of a commercial product/device. Adenoviruses are common, accounting for at least 5%–10% of respiratory infections in children.1 Vomiting and diarrhea are often present concurrently.2 A specific viral etiology is usually not sought in children with these self-limited symptoms, so the incidence of adenovirus infections is difficult to ascertain. In the current study, the authors reviewed rare CNS manifestations of... You do not currently have access to this content.

2048. Comparison Between Endpoint and Real-Time (RT) Polymerase Chain Reaction (PCR) for the Diagnosis of Pneumocystis Pneumonia (PCP)

BackgroundThe definitive diagnosis of PCP requires direct visualization of the organism by silver or direct fluorescent antibody stain, but in recent years PCR has become a widely used diagnostic tool. Varying results have been noted with different PCR assays; one concern has been that RT-PCR will be more sensitive and not differentiate colonization from infection. For this study, we compared the performance of RT-PCR with that of endpoint PCR for detection of PCP.MethodsAll adult patients who had a bronchoalveolar lavage (BAL) or sputum sample positive for Pneumocystis by PCR at the U. Michigan Hospitals from February 2014–February 2018 were studied. Before February 2017 samples were tested with endpoint PCR followed by agarose gel electrophoresis and after February 2017 with RT-PCR. For each patient, a strict case definition based on host factors, clinical presentation, radiological and pathologic findings, was used to classify PCP as proven, probable, possible, and unlikely. Based on this classification, endpoint PCR and RT-PCR results were designated as true positive or false-positive presumably colonized (FP).ResultsThe number of specimens tested each year was similar, ranging from 751 to 791. One hundred and fifty-three patients tested positive: 77/2318 (3%) by endpoint PCR and 76/783 (10%) by RT-PCR. One hundred and twenty-six patients had risk factors for PCP: hi-dose steroids (39), hematologic malignancy (38), chemotherapy within 3 months (24), HIV (14), solid-organ transplant (12), stem cell transplant (9), and 27 patients had no PCP risk factors. By our definitions, patients were classified as proven (2), probable (70), possible (46) and unlikely (35). RT-PCR gave a higher FP rate (27/76, 35%) than endpoint PCR (8/77, 10%,) P < 0.0001, especially in those with chronic lung disease, P = .001 and those with no known PCP risk factors, P < 0.0001. More patients with no risk factors tested positive with RT-PCR (20) than with endpoint PCR (7), P = .006. FP rates RT-PCR were similar in sputum (34%) and BAL (36%).ConclusionRT-PCR gave significantly more FP results, likely due to increased detection of Pneumocystis colonization. Pretest probability should be considered when ordering a highly sensitive test such as RT-PCR and positive results must be interpreted in the context of the clinical presentation, radiological findings and risk factors.Disclosures All authors: No reported disclosures.

Adenovirus respiratory tract infections in infants: a retrospective chart-review study

BackgroundHuman adenoviruses have an important role in paediatric respiratory tract infections. They are estimated to cause 2–5% of the overall respiratory tract infections and 4–10% of all pneumonias. The aim of this study was to evaluate the clinical presentation and effect of adenoviral infection on the management of infected infants. MethodsData were collected from the medical records of patients infected with adenovirus and admitted to Caritas Baby Hospital. Adenoviral respiratory tract infections were diagnosed from nasopharyngeal aspirates using direct fluorescent antibody staining. We analysed patient clinical presentation, medical workup, laboratory workup, and antibiotic administration. This study was approved by Caritas Baby Hospital Medical Research Committee. FindingsWe reviewed records for 491 patients admitted to Caritas Baby Hospital with adenoviral infection between Jan 1, 2006, and June 30, 2016. Adenoviral activity was noted throughout the months of the study period, with major activity during late winter, spring, and early summer. Boys were most affected (male to female ratio 2:1). Upon admission, 187 (38%) patients were afebrile. According to the clinical presentation, 327 (67%) patients presented with upper respiratory tract infection symptoms, 165 (34%) with gastrointestinal tract symptoms, 59 (12%) with difficulty of breathing, and 46 (9%) with conjunctivitis. 279 (57%) patients had leucocytosis, whereas the C-reactive protein titre was more than 50 μg/mL in 228 (46%) patients. 92 (19%) patients needed a lumbar puncture. Overall, 354 (72%) patients received antibiotic treatment. Length of hospital stay was 1–10 days, and most patients were discharged from hospital on day 3. The average cost of hospitalisation was US$1180·5 per patient. InterpretationAdenoviral infections in infants can present with a sepsis-like picture, mandating unnecessary interventions. Clinical laboratories in hospitals must therefore have rapid and sensitive adenovirus detection techniques to assist the doctors in making appropriate treatment decisions. FundingMedical Research Committee of Caritas Baby Hospital.

The 2015 New York City Legionnaires' Disease Outbreak: A Case Study on a History-Making Outbreak.

The 2015 New York City Legionnaires' Disease Outbreak: A Case Study on a History-Making Outbreak.

Accuracy of diagnostic tests for Legionnaires' disease: a systematic review.

Rapid and effective diagnosis of Legionnaires' disease (LD) cases is extremely important so that timely and appropriate therapy can be provided, thereby lowering the morbidity and mortality rates and reducing the health and economic costs associated with this disease. Diagnosis is established solely by microbiological tests. There are several methods available, each with different performance, sensitivity and specificity characteristics, and further understanding is required. Our objective was to assess the accuracy of urinary antigen detection, direct fluorescent antibody (DFA) staining, serological testing and the polymerase chain reaction (PCR) method versus culture analysis (the reference standard) in patients suspected of being infected with Legionella or patients with laboratory-confirmed LD. We performed a MEDLINE search in November 2014. Two authors independently assessed the trials and extracted data. Pooled analysis was performed through Meta-DiSc version 1.4. The inclusion criteria were met by 11 studies. All the studies evaluated PCR and DFA tests to detect Legionella in clinical specimens, comparing them to culture techniques, and were included in the meta-analysis. The pooled sensitivity and specificity for PCR were 83 % [95 % confidence interval (CI): 79-87 %] and 90 % (95 % CI: 88-92 %), respectively. DFA was evaluated in one study and the sensitivity and specificity of this test were 67 % (95 % CI: 30-93 %) and 100 % (95 % CI: 91-100 %), respectively. PCR had high sensitivity and specificity for early diagnosis of LD. Culture analysis is deemed necessary for epidemiological studies, molecular strain typing and antibiotic sensibility evaluations; however, the performance of PCR in recent studies calls for additional, well-designed studies in order to achieve the best standard test, which will enable optimization of the Legionella infection diagnostic.

Lower respiratory tract viral infections: Diagnostic role of exfoliative cytology.

Viral lower respiratory tract infections (VLRTI) remain one of the most common causes of morbidity and mortality worldwide. For many years, the diagnosis of VLRTI was based on laboratory techniques such as viral isolation in cell culture, antigen detection by direct fluorescent antibody staining, and rapid enzyme immunoassay. Radiological imaging and morphology also play an important role in diagnosing these infections. Exfoliative cytology provides a simple, rapid, inexpensive, and valuable means to diagnose and manage VLRTI. Here we review viral‐associated cytomorphological changes seen in exfoliated cells of the lower respiratory tract. Diagn. Cytopathol. 2017;45:614–620. © 2017 Wiley Periodicals, Inc.

Non-dermatomal varicella-zoster skin infection: disseminated cutaneous herpes zoster without dermatome in an immunosuppressed woman

Disseminated herpes zoster is defined as the presence of more than 20 lesions outside the dermatome. This unusual presentation is more common in immunosuppressed patients. Complications such as hepatitis, encephalitis, and pneumonitis are more likely in individuals with disseminated varicella zoster virus infection.A 63-year-old woman being treated for breast cancer developed multiple pustules and vesicles days after starting doxorubicin and cyclophosphamide chemotherapy. Ten individual lesions appeared on her chest, abdomen, back, and leg. Non-dermatomal disseminated herpes zoster was suspected. She was treated with oral antiviral therapy, as well as with oral and topical antibiotics. Varicella zoster virus infection was confirmed by direct fluorescent antibody staining. After one month, her skin lesions had resolved and she resumed chemotherapy.In a setting of immunosuppression, the rare presentation of disseminated herpes zoster without dermatome should be considered. Appropriate antiviral therapy should be administered while waiting for confirmation of the diagnosis, so as to reduce the risk of visceral dissemination of the varicella zoster virus infection.

Atypical Pneumocystis jirovecii pneumonia in previously untreated patients with CLL on single-agent ibrutinib

AbstractIbrutinib is not known to confer risk for Pneumocystis jirovecii pneumonia (PCP). We observed 5 cases of PCP in 96 patients receiving single-agent ibrutinib, including 4 previously untreated. Clinical presentations included asymptomatic pulmonary infiltrates, chronic cough, and shortness of breath. The diagnosis was often delayed. Median time from starting ibrutinib to occurrence of PCP was 6 months (range, 2-24). The estimated incidence of PCP was 2.05 cases per 100 patient-years (95% confidence interval, 0.67-4.79). At the time of PCP, all patients had CD4 T-cell count >500/μL (median, 966/μL) and immunoglobulin G (IgG) >500 mg/dL (median, 727 mg/dL). All patients underwent bronchoalveolar lavage. P jirovecii was identified by polymerase chain reaction in all 5 cases; direct fluorescence antibody staining was positive in 1. All events were grade ≤2 and resolved with oral therapy. Secondary prophylaxis was not given to 3 patients; after 61 patient-months of follow up, no recurrence occurred. Lack of correlation with CD4 count and IgG level suggests that susceptibility to PCP may be linked to Bruton tyrosine kinase (BTK) inhibition. If confirmed, this association could result in significant changes in surveillance and/or prophylaxis, possibly extending to other BTK inhibitors. This trial was registered at www.clinicaltrials.gov as #NCT01500733 and #NCT02514083.

Profile of pneumocystis infection in a tertiary care institute in North India

Context:Pneumocystis jirovecii pneumonia (PcP) is still remains a common opportunistic disease in human immunodeficiency virus (HIV) infected individuals. Study on PcP in developing countries are scarce.Aims:To study the occurrence of P. jirovecii infection in clinically suspected individuals in a tertiary care institute.Settings and Design:Retrospective study conducted in a tertiary care hospital.Materials and Methods:Two years data regarding respiratory sample analysis, HIV status, and cluster of differentiation 4 (CD4) cell count of clinically suspected pneumocystis infection patients with known/unknown HIV status were analyzed.Results:Data of 45 eligible patients were analyzed. The majority of the patients were male (between 21 and 50 years of age). Total 26 (57.7%) patients were HIV reactive, of which 14 had CD4 count of <200 cells/mm3. 20 patients (9 HIV reactive and 11 unknown HIV status) were confirmed with pneumocystosis by direct fluorescent antibody (DFA) staining. Four of 14 HIV reactive individuals who had CD4 count of <200 cells/mm3 and 5 of 12 HIV reactive individuals who had CD4 count of >200 cells/mm3 were positive for pneumocystosis.Conclusions:Pneumocystis pneumonia is still prevalent in North India and is mainly affecting patients in economically productive and sexually active age group. To diagnose pneumocystosis, DFA is an easily available method in resource-limited settings. Appreciating the actual HIV or immunodeficiency status and the CD4 profile of an individual with symptoms of pneumocystis infection will help the clinicians in early diagnosis and initiation appropriate therapy in individuals living with the disease.

Profile of pneumocystis infection in a tertiary care institute in North India.

Pneumocystis jirovecii pneumonia (PcP) is still remains a common opportunistic disease in human immunodeficiency virus (HIV) infected individuals. Study on PcP in developing countries are scarce. To study the occurrence of P. jirovecii infection in clinically suspected individuals in a tertiary care institute. Retrospective study conducted in a tertiary care hospital. Two years data regarding respiratory sample analysis, HIV status, and cluster of differentiation 4 (CD4) cell count of clinically suspected pneumocystis infection patients with known/unknown HIV status were analyzed. Data of 45 eligible patients were analyzed. The majority of the patients were male (between 21 and 50 years of age). Total 26 (57.7%) patients were HIV reactive, of which 14 had CD4 count of <200 cells/mm3. 20 patients (9 HIV reactive and 11 unknown HIV status) were confirmed with pneumocystosis by direct fluorescent antibody (DFA) staining. Four of 14 HIV reactive individuals who had CD4 count of <200 cells/mm3 and 5 of 12 HIV reactive individuals who had CD4 count of >200 cells/mm3 were positive for pneumocystosis. Pneumocystis pneumonia is still prevalent in North India and is mainly affecting patients in economically productive and sexually active age group. To diagnose pneumocystosis, DFA is an easily available method in resource-limited settings. Appreciating the actual HIV or immunodeficiency status and the CD4 profile of an individual with symptoms of pneumocystis infection will help the clinicians in early diagnosis and initiation appropriate therapy in individuals living with the disease.

Detection of Respiratory Syncytial Virus using Direct Fluorescent Antibody Assay in Paediatric Patients with Acute Respiratory Tract Infection.

Severe Respiratory Syncytial Virus (RSV) pulmonary disease manifesting as bronchiolitis and pneumonia continues to play a major role in the childhood mortality and morbidity. Hence the present study was undertaken to evaluate the prevalence of RSV among hospitalized children presenting with Acute Respiratory Tract Infection (ARTI) and its correlation with risk factors. To determine the occurrence of RSV related respiratory tract infection in paediatric patients and to access the risk factors and clinical features associated. RSV antigen detection was performed by Direct Fluorescent Antibody (DFA) staining on 100 nasopharyngeal aspirate collected from hospitalized children below 5 years of age with a diagnosis of ARTI. Out of the 100 samples tested for RSV with DFA, 22 (22%) were found RSV positive with a mean age of 12 months and a male to female ratio of (1.75:1). Clinical features significantly associated with RSV were wheezing and breathlessness. Congenital heart disease (CHD) and prematurity were the risk factors significantly associated with RSV infection. RSV infection is a significant cause of morbidity among children presenting with ARTI. In resource limited countries DFA can be used as an important tool for rapid detection of RSV and can potentially eliminate prolonged hospitalization and unnecessary use of antibiotics.

Diagnostic value of direct fluorescence antibody staining for detecting Pneumocystis jirovecii in expectorated sputum from patients with HIV infection

Direct fluorescent antibody (DFA) staining of induced sputum is frequently used to diagnose Pneumocystis pneumonia (PCP) in patients infected with human immunodeficiency virus, although induction can provoke nausea and bronchospasm. Since the diagnostic value of expectorated sputum examined with DFA stain has not been well evaluated, we reviewed the medical records of HIV-infected patients who were clinically diagnosed as having PCP between 1999 and 2011. Over this 13-year period, we found 76 patients whose records included the results of DFA staining of expectorated sputum and noted that 42 (55.3%) were positive. Polymerase chain reaction to detect Pneumocystis in the sputum of 65 of the patients resulted in the finding of 43 (66.2%) who were positive. Our findings suggest that DFA staining of expectorated sputum could be a useful initial diagnostic method in HIV-infected patients with PCP.

Multiplex Viral Polymerase Chain Reaction Testing Using the FilmArray Device Compared With Direct Fluorescent Antibody Testing

Advances in fluidics, electronics, and instrument design have enabled automation of complex molecular assays. The FilmArray Respiratory Panel (RP) is one such assay; it allows for rapid detection of multiple viral and bacterial respiratory targets via nested polymerase chain reaction (PCR), with all components contained within a single pouch. We performed a detailed comparison of workflow between the FilmArray RP and direct fluorescent antibody (DFA) staining. The FilmArray RP proved to be more efficient, with as few as 91 total touches needed and as little as 4 minutes 52 seconds hands-on time. This compares with as many as 502 total touches and 12 minutes 45 seconds hands-on time for DFA staining. FilmArray RP detected a greater number of organisms (20 viruses and bacteria vs 7 viruses for DFA staining) and required less training compared with DFA staining. The ease of use of the FilmArray RP makes this molecular assay amenable to operation 24 hours a day, 7 days a week in routine laboratory and point-of-care settings.

Detection of asymptomatic renal Leptospira infection in abattoir slaughtered cattle in southeastern Georgia, United States.

Objectives:Leptospirosis is one of the most widespread zoonotic infectious diseases affecting humans and animals. Several animal species, including cattle, can act as potential asymptomatic carriers facilitating zoonotic transmission of Leptospira. This study was conducted to assess the occurrence of asymptomatic renal Leptospira carriers among cattle slaughtered in southeastern Georgia, United States.Methods:A battery of diagnostic tests, including dark field microscopy, direct fluorescent antibody staining, polymerase chain reaction, and culture, were performed on a set of bovine kidneys (n = 37) collected from an abattoir in southeastern Georgia, United States. Virulence of a field isolate obtained from this study was tested in a hamster experimental model.Results:Motile spirochete-like structures were observed by dark field microscopy in 23 (59%) out of 37 kidney samples tested. In all, 29 samples (78%) were positive by direct fluorescent antibody staining. Only 11 (29.7%) samples by polymerase chain reaction and 3 (8.1%) by culture were positive for Leptospira sp. The isolates obtained by culture were confirmed as Leptospira borgpetersenii. Hamsters experimentally infected with one of the Leptospira field isolates obtained from this study did not show clinical signs but developed renal infection with interstitial nephritis and tubular necrosis.Conclusions:This study confirms that asymptomatic Leptospira renal infection is present among cattle in the region. Our findings underscore the need for future studies to assess the potential environmental contamination and transmission to humans in contact with infected cattle.

Simultaneous detection of Giardia lamblia and Cryptosporidium parvum (oo)cysts in soil using immunomagnetic separation and direct fluorescent antibody staining

An improved approach for simultaneous detection of Cryptosporidium parvum and Giardia lamblia (oo)cysts in soil is described. Recoveries>70% were obtained for concentrations>55 and 21 (oo)cysts g−1 for C. parvum and G. lamblia, respectively. The limits of detection were determined to be<5 (oo)cysts g−1 soil.

Investigation of Chlamydia trachomatis with Cell Culture, DFA and PCR Methods in the Genital Swab Samples of Symptomatic Patients

Chlamydia trachomatis infection is considered the most prevalent bacterial sexually transmitted disease worldwide. C.trachomatis causes eye infections such as trachoma and newborn inclusion conjunctivitis, newborn pneumonia, genitourinary system infections and suppurative inguinal lymphadenitis namely lymphogranuloma venerum. The aim of this study was to investigate C.trachomatis by direct fluorescent antibody (DFA), polymerase chain reaction (PCR) and cell culture methods in the clinical samples sent to the microbiology laboratory with the prediagnosis of genital infections. A total of 50 swab samples obtained from adult patients (49 female, 1 male) who were admitted to Erciyes University Hospital, Kayseri, Turkey between February-March 2010, were included in the study. C.trachomatis antigens were investigated by a commercial DFA (PathoDx, Remel, USA) method. McCoy cell cultures prepared in microplate wells were used for the isolation of C.trachomatis. The growth of C.trachomatis in cell cultures was confirmed by DFA and iodine staining methods. C.trachomatis DNA was investigated by commercially available PCR (Chlamydia trachomatis 330/740 IC; Sacace, Italy) method. In our study, 4 (8%) of the 50 swab samples were found positive with DFA, 1 (2%) was positive with cell culture, and 1 (2%) was positive with PCR. The only sample that gave positive results with all of the three methods was an urethral swab. Three cervical swab samples that were found positive only with DFA method was evaluated as false positivity. When cell culture was considered as the reference method, the sensitivity and specificity of DFA method were estimated as 100% and 94%, respectively, while those rates for PCR were 100% and 100%, respectively. In conclusion, although cell culture is still the gold standard in the diagnosis of C.trachomatis. infections, since it is time consuming and difficult to apply, more rapid and reliable PCR methods may be applied in diagnosis. DFA method which is practical and cheap, is preferred largely in routine laboratory practice. However, false negative and false positive DFA results should be prevented by the maintainence of good quality clinical specimens, evaluation of the test by experienced personnel and use of quality control samples in each run.