Detection of Specific Antibodies to an Antigenic Mannoprotein for Diagnosis of Penicillium marneffei Penicilliosis

The disseminated and progressive fungal disease Penicillium marneffei penicilliosis is one of the most common infectious diseases in AIDS patients in Southeast Asia. To diagnose systemic penicilliosis, we developed an enzyme-linked immunosorbent assay (ELISA)-based antibody test with Mp1p, a purified recombinant antigenic mannoprotein of P. marneffei. Evaluation of the test with guinea pig sera against P. marneffei and other pathogenic fungi indicated that this assay was specific for P. marneffei. Clinical evaluation revealed that high levels of specific antibody were detected in two immunocompetent penicilliosis patients. Furthermore, approximately 80% (14 of 17) of the documented penicilliosis patients with human immunodeficiency virus tested positive for the specific antibody. No false-positive results were found for serum samples from 90 healthy blood donors, 20 patients with typhoid fever, and 55 patients with tuberculosis, indicating a high specificity of the test. Thus, this ELISA-based test for the detection of anti-Mp1p antibody can be of significant value as a diagnostic for penicilliosis.     

Identification and Purification of Specific Penicillium marneffei Antigens and Their Recognition by Human Immune Sera

Disseminated infection with the dimorphic pathogenic fungus Penicillium marneffei is increasingly seen among patients with AIDS in southeast Asian countries. Previous studies have demonstrated the presence of humoral immune responses to this fungus in patient sera; we have confirmed this work using sera from P. marneffei-infected patients (n = 21) to develop Western blots of P. marneffei cytoplasmic yeast antigen (CYA). P. marneffei CYA was then partially purified by liquid isoelectric focusing, and fractions were subjected to sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and Western blotting. Immunoenzyme development of the Western blots with pooled sera from patients with P. marneffei infection and with pooled sera from patients with aspergillosis (n = 20), candidiasis (n = 10), cryptococcosis (n = 9), and histoplasmosis (n = 11) revealed three antigens with relative molecular masses of 61, 54, and 50 kDa. These antigens were specifically recognized by the pooled sera from the P. marneffei-infected patients. The 61- and 54-kDa antigens were subsequently purified to homogeneity by preparative gel electrophoresis, and the 50-kDa antigen was partially purified by the same technique. N-terminal amino acid sequencing revealed that the 61-kDa antigen had a strong homology (87% identity) with the antioxidant enzyme catalase. The three antigens were then subjected to SDS-PAGE and Western blotting and to immunoenzyme development with individual patient sera; sera from 86% of P. marneffei-infected patients recognized the 61-kDa antigen, sera from 71% recognized the 54-kDa antigen, and sera from 48% recognized the 50-kDa antigen. These specifically recognized antigens are the first to be purified from P. marneffei and can be used either singly or in combination to detect antibody responses in a large percentage of individuals infected with P. marneffei.     

Detection of circulating galactomannan in serum samples for diagnosis of Penicillium marneffei infection and cryptococcosis among patients infected with human immunodeficiency virus

Galactomannan (GM) is a heteropolysaccharide in the cell walls of most Aspergillus and Penicillium species. Cross-reactivity of Cryptococcus neoformans galactoxylomannan in an Aspergillus GM test has also been reported. In this study, we used a Platelia Aspergillus enzyme immunoassay kit (Bio-Rad) to test serum samples obtained from 48 human immunodeficiency virus (HIV)-infected patients (15 with penicilliosis [7 with fungemia alone, 4 with cavitary lung lesions alone, 3 with both fungemia and cavitary lung lesions, and 1 with disseminated disease], 22 with cryptococcosis [11 with fungemia alone, 5 with cavitary lung lesions, 3 with both, and 3 with meningitis alone], and 11 without any invasive fungal infection [control]) for GM levels. None of the patients had aspergillosis or concurrent use of piperacillin-tazobactam or amoxicillin-clavulanate. The median time between diagnosis of fungal infection and collection of serum samples was 0 days for penicilliosis and 1.5 days for cryptococcosis. Of patients with penicilliosis, cryptococcosis, and controls, 73.3%, 13.6%, and 9%, respectively, had GM optical density (OD) indices of >0.5 (P = 0.0001). GM OD indices were higher for penicilliosis (median OD index, 4.419; range, 0.158 to >20) than for cryptococcosis (median, 0.247; range, 0.112 to 3.849) cases (P < 0.001). Patients with fungemic penicilliosis had higher OD indices (median, 10.628; range, 0.401 to >20) than patients with nonfungemic penicilliosis (median, 0.378; range, 0.158 to 4.419) and patients with cryptococcemia (median, 0.231; range, 0.112 to 1.168) (P < 0.001). Of the 15 patients with cavitary lung lesions, those with penicilliosis had higher antigen levels (median OD index, 1.641; range, 0.247 to >20) than those with cryptococcosis (median, 0.227; range, 0.112 to 3.849) (P = 0.011). This study showed that the GM OD index was significantly elevated for HIV patients with penicilliosis. The use of the GM antigen assay may facilitate earlier diagnosis of Penicillium marneffei infection for HIV-infected patients in areas of endemicity.     

Development and evaluation of rapid urinary antigen detection tests for diagnosis of penicilliosis marneffei

Penicilliosis, caused by the dimorphic fungus Penicillium marneffei, is an important opportunistic systemic fungal infection affecting immunocompromised individuals living in areas where penicilliosis is endemic. We have demonstrated previously that a urinary enzyme-linked immunosorbent assay (ELISA) with purified rabbit polyclonal antibody against killed whole-fission-form arthroconidia of P. marneffei was specific and highly sensitive for the diagnosis of penicilliosis. In this study, a dot blot ELISA and a latex agglutination (LA) test were developed with the same polyclonal antibody and compared with the ELISA for the detection of P. marneffei urinary antigen. Urine specimens from 37 patients with culture-proven penicilliosis and 300 controls (52 healthy subjects and 248 hospitalized patients without penicilliosis) were tested. Antigen was detected in urine from all 37 (100%) penicilliosis patients by the LA test, 35 (94.6%) penicilliosis patients by the dot blot ELISA, and 36 (97.3%) penicilliosis patients by the ELISA. False-positive results were found by the three assays for 2 (0.7%), 8 (2.7%), and 6 (2%) of 300 controls, respectively. The overall sensitivities of the diagnostic tests were as follows: dot blot ELISA, 94.6%; ELISA, 97.3%; and LA test, 100% (specificities, 97.3, 98, and 99.3%, respectively). The LA test is simple, robust, rapid, and convenient and should prove to be an important addition to the existing diagnostic tests for penicilliosis.     

Detection of histoplasma antigen by a quantitative enzyme immunoassay

The second-generation Histoplasma antigen immunoassay is semiquantitative, expressing results as a comparison to a negative control, which requires repeat testing of the prior specimen with the current specimen to accurately determine a change in antigen. Reporting results in this manner often is confusing to the ordering physician and laboratory. Development of a quantitative assay could improve accuracy, reduce interassay variability, and eliminate the need to test the prior sample with the current sample in the same assay. Calibrators with known concentrations of Histoplasma antigen were used to quantitate antigen in specimens from patients with histoplasmosis and from controls. Samples from cases of disseminated histoplasmosis or other mycoses and controls were tested to evaluate the performance characteristics of the quantitative assay. Paired specimens were evaluated to determine if quantitation eliminated the need to test the current and prior specimens in the same assay to assess a change in antigen. The sensitivity in samples from patients with AIDS and disseminated histoplasmosis was 100% in urine and 92.3% in serum. Cross-reactions occurred in 70% of other endemic mycoses, but not in aspergillosis. Specificity was 99% in controls with community-acquired pneumonia, medical conditions in which histoplasmosis was excluded, or healthy subjects. A change in antigen level categorized as an increase, no change, or decrease based on antigen units determined in the same assay agreed closely with the category of change in nanograms/milliliter determined from testing current and prior specimens in different assays. Sensitivity, specificity, and interassay precision are excellent in the new third-generation quantitative Histoplasma antigen immunoassay.     

A novel inhibition ELISA for the detection and monitoring of <Emphasis Type="Italic">Penicillium marneffei</Emphasis> antigen in human serum

The thermally dimorphic fungus Penicillium marneffei is a causative agent of penicilliosis marneffei, a disease considered to be an acquired immune deficiency syndrome (AIDS)-defining illness in Southeast Asia and southern China. We have developed an inhibition enzyme-linked immunosorbent assay (inh-ELISA) incorporating the yeast phase specific mannoprotein-binding monoclonal antibody 4D1 for the detection of P. marneffei infection. In our sample set, the test detected antigenemia in all 45 (100 %) patients with P. marneffei, with a mean antigen concentration of 4.32 μg/ml. No cross-reactivity in this assay was found using serum from 44 additional patients with other fungal infections, such as Aspergillus fumigatus, Cryptococcus neoformans, and Candida albicans, as well as 44 patients with bacterial infections, such as Mycobacterium tuberculosis and Streptococcus suis. Additionally, no reactivity occurred using serum from 31 human immunodeficiency virus (HIV)-infected patients without a history of fungal infections and 113 healthy controls residing in endemic areas. To investigate the potential of the inh-ELISA for disease monitoring, we followed the reduction in antigenemia in six patients who clinically responded to itraconazole and P. marneffei was no longer isolated from their blood or tissues. In contrast, we correlated increased concentrations of antigenemia in patients with relapsed P. marneffei infection with the progression of their clinical symptoms and the isolation of P. marneffei from their clinical specimens. In summary, the P. marneffei inh-ELISA is a promising new assay for the rapid diagnosis of P. marneffei, as well as a tool for evaluating clinical response and clearance of the fungus during treatment.     

Value of Serial Quantification of Fungal DNA by a Real-Time PCR-Based Technique for Early Diagnosis of Invasive Aspergillosis in Patients with Febrile Neutropenia

A study was designed to assess the reliability of the serial detection of Aspergillus sp. DNA to diagnose invasive aspergillosis (IA) in patients with febrile neutropenia. Two blood and two serum samples were taken weekly from 83 patients. A total of 2,244 samples were analyzed by real-time quantitative PCR. Twelve (14.4%) patients were diagnosed with IA. Taking two consecutive positive results as the diagnostic criterion, PCR detected 11 cases, with 4 false positives, giving sensitivity, specificity, positive, and negative predictive values of 91.6%, 94.4%, 73.3%, and 98.5%, respectively. On analyzing in conjunction with high-resolution chest tomography (HRCT) and galactomannan (GM) testing, the combination of serial PCR and GM detected 100% of aspergillosis cases, with a positive predictive value of 75.1%. This diagnostic strategy presented, according to CART analysis, a receiver-operator curve with an area under the curve of 0.97 (95% confidence interval, 0.895 to 1.032; P < 0.01), with a relative risk of IA 6.92 times higher than the control population and with predictive success of 95.2%. As regards early diagnosis, the serial detection of Aspergillus DNA took on average 21 days less than HRCT and 68 days less than GM. The serial detection of Aspergillus DNA using real-time quantitative PCR has great diagnostic applicability, which increases when combined with GM quantification.     

Rapid diagnosis of vivax malaria by the SD Bioline Malaria Antigen test when thrombocytopenia is present

An easy and reliable diagnostic method for malaria is highly desirable. We examined the recently introduced SD Bioline Malaria Antigen test, which detects Plasmodium lactate dehydrogenase, with the additional aid of the presence or absence of thrombocytopenia to diagnose vivax malaria. We enrolled 732 patients with clinically suspected malaria in an area where vivax malaria is endemic. We performed microscopic examination of thin film, applied the SD Bioline Malaria Antigen test, and checked platelet counts. One hundred ninety-five patients were smear positive for vivax malaria. The sensitivity of the SD Bioline Malaria Antigen test was 96.4%, and its specificity was 98.9%. We found that 95.4% of malaria patients had thrombocytopenia, and the proportion with malaria increased as platelet counts decreased. A positive SD Bioline Malaria Antigen test when thrombocytopenia was present showed a 100% positive predictive value for vivax malaria. In conclusion, the SD Bioline Malaria Antigen test is a rapid and accurate diagnostic method for vivax malaria, and a platelet count can facilitate a rapid diagnosis of malaria.     

Utility of real-time PCR for diagnosis of Legionnaires' disease in routine clinical practice

The main aim of our study was to determine the added value of PCR for the diagnosis of Legionnaires’ disease (LD) in routine clinical practice. The specimens were samples submitted for routine diagnosis of pneumonia from December 2002 to November 2005. Patients were evaluated if, in addition to PCR, the results of at least one of the following diagnostic tests were available: (i) culture for Legionella spp. on buffered charcoal yeast extract agar or (ii) detection of Legionella pneumophila antigen in urine specimens. Of the 151 evaluated patients, 37 (25%) fulfilled the European Working Group on Legionella Infections criteria for a confirmed case of LD (the “gold standard”). An estimated sensitivity, specificity, and overall percent agreement of 86% (32 of 37; 95% confidence interval [CI] = 72 to 95%), 95% (107 of 112; 95% CI = 90 to 98%), and 93% (139 of 149), respectively, were found for 16S rRNA-based PCR, and corresponding values of 92% (34 of 37; 95% CI = 78 to 98%), 98% (110 of 112; 95% CI = 93 to 100%), and 97% (144 of 149), respectively, were found for the mip gene-based PCR. A total of 35 patients were diagnosed by using the urinary antigen test, and 34 were diagnosed by the 16S rRNA-based PCR. With the mip gene PCR one more case of LD (n = 36; not significant) was detected. By combining urinary antigen test and the mip gene PCR, LD was diagnosed in an additional 4 (11%) patients versus the use of the urinary antigen test alone. The addition of a L. pneumophila-specific mip gene PCR to a urinary antigen test is useful in patients with suspected LD who produce sputum and might allow the early detection of a significant number of additional patients.     

Sensitivity of Fluorochrome Microscopy for Detection of Mycobacterium tuberculosis versus Nontuberculous Mycobacteria

The results for 6,532 consecutive mycobacterial respiratory specimens collected from 1,040 patients from 1993 to 1995 in a Texas hospital were studied to determine the sensitivity of fluorescence microscopy for detection of Mycobacterium tuberculosis and nontuberculous mycobacteria (NTM). Smears were positive for acid-fast bacilli (AFB) in 63% (677 of 1,082) of specimens growing M. tuberculosis and 56% (638 of 1,148) of specimens growing the four most common species of NTM. Smear positivity by species was 58% (446 of 776) for M. avium complex, 51% (154 of 300) for rapidly growing mycobacteria (98% were M. abscessus), 78% (29 of 37) for M. kansasii, and 26% (9 of 35) for M. gordonae. Definite or probable disease by clinical criteria was present in 79% of patients with M. avium complex, 93% of patients with rapidly growing mycobacteria, 100% of patients with M. kansasii, and 0% of patients with M. gordonae. Patients with M. avium complex had a low incidence of AIDS (7%), and approximately 50% of non-AIDS patients had upper-lobe cavitary disease and 50% had nodular bronchiectasis. Only 23 of 6,532 (0.35%) of AFB smears were positive with a negative culture excluding patients on therapy for established mycobacterial disease. These studies suggest that NTM are as likely as M. tuberculosis to be detected by fluorescent microscopy in specimens from patients from areas endemic for NTM lung disease and at low risk for AIDS.     

Rapid Detection of a Schistosoma mansoni Circulating Antigen Excreted in Urine of Infected Individuals by Using a Monoclonal Antibody

Schistosoma circulating antigens were used to indicate the infection intensity and to assess cure. An immunoglobulin G2a (IgG2a) mouse monoclonal antibody was used in a fast dot-enzyme-linked immunosorbent assay (ELISA; FDA) for rapid and simple diagnosis of schistosomiasis in the field. Seven hundred Egyptians were parasitologically examined for Schistosoma mansoni and other parasitic infections. A rectal biopsy was done as a “gold standard” for individuals showing no S. mansoni eggs in their feces. Egg counts were obtained by the Kato smear method for only 100 of 152 individuals with eggs in their feces. Specific anti-schistosome IgG antibodies were evaluated in sera by ELISA. Urine samples from the 700 individuals were tested by FDA for detection of the circulating antigen. The assay showed a sensitivity of 93% among 433 infected individuals and a specificity of 89% among 267 noninfected individuals. FDA showed the highest efficiency of antigen detection (91%) compared with the efficiency of antibody detection by ELISA (75%) and stool analysis (60%). In addition, FDA detected infected patients with 20 eggs/g of feces. Also, the sensitivity of FDA ranged from 90 to 94% among samples from patients with different clinical stages of schistosomiasis. All the assay steps can be completed within 30 min at room temperature for 96 urine samples. The monoclonal antibody identified a 74-kDa antigen in different antigenic extracts of S. mansoni and Schistosoma haematobium and in the urine of infected individuals. In addition, a 30-kDa degradation product was identified only in the urine samples. On the basis of these results, FDA should be used as a rapid tool for the sensitive and specific diagnosis of Schistosoma infection.     

Comparison of Commercial Latex Agglutination and Sandwich Enzyme Immunoassays with a Competitive Binding Inhibition Enzyme Immunoassay for Detection of Antigenemia and Antigenuria in a Rabbit Model of Invasive Aspergillosis

A commercial latex agglutination assay (LA) and a sandwich enzyme immunoassay (SEIA) (Sanofi Diagnostics Pasteur, Marnes-la-Coquette, France) were compared with a competitive binding inhibition assay (enzyme immunoassay [EIA]) to determine the potential uses and limitations of these antigen detection tests for the sensitive, specific, and rapid diagnosis of invasive aspergillosis (IA). Toward this end, well-characterized serum and urine specimens were obtained by using a rabbit model of IA. Serially collected serum or urine specimens were obtained daily from control rabbits or from rabbits immunosuppressed and infected systemically with Aspergillus fumigatus. By 4 days after infection, EIA, LA, and SEIA detected antigen in the sera of 93, 93, and 100% of A. fumigatus-infected rabbits, respectively, whereas antigen was detected in the urine of 93, 100, and 100% of the rabbits, respectively. False-positive results for non-A. fumigatus-infected rabbits for EIA, LA, and SEIA were as follows: for serum, 14, 11, and 23%, respectively; for urine, 14, 84, and 90%, respectively. Therefore, although the sensitivities of all three tests were similar, the specificity was generally greater for EIA than for LA or SEIA. Infection was also detected earlier by EIA, by which the serum of 53% of A. fumigatus-infected rabbits was positive as early as 1 day after infection, whereas the serum of only 27% of the rabbits tested by LA was positive. Although the serum of 92% of A. fumigatus-infected rabbits was positive by SEIA as early as 1 day after infection, the serum of a high percentage (50%) was false positive before infection. The urine of 21% of A. fumigatus-infected rabbits was positive by EIA as early as 1 day after infection, and the urine of none of the rabbits was false positive before infection. When EIA results for urine specimens were combined with those for serum, sensitivity was improved (i.e., 67% of rabbits were positive by 1 day after infection and only one rabbit gave a false-positive result). A total of 93% of A. fumigatus-infected rabbits were positive for antigen in urine as early as 1 day after infection and the urine of 100% of the rabbits was positive by SEIA. However, before infection, 79% of A. fumigatus-infected rabbits were false positive for antigen in urine by LA and 90% were false positive for antigen in urine by SEIA. These data indicate that the EIA has the potential to be used to diagnose IA with both serum and urine specimens and to detect a greater number of infections earlier with greater specificity than the specificities achieved with the commercial tests.     

Candida tropicalis and Penicillium marneffei Mixed Fungaemia in a Patient with Waldenström's Macroglobulinaemia

A patient with Waldenström's macroglobulinaemia who had previously been treated with cladribine presented with septic arthritis caused by Escherichia coli. The patient's condition subsequently deteriorated, and he succumbed to pneumonia and mixed fungaemia due to Candida tropicalis and Penicillium marneffei. Profound lymphopenia coexisted with fungaemia. This is the first reported case of mixed Penicillium marneffei and Candida fungaemia and penicilliosis marneffei in a patient with Waldenström's macroglobulinaemia. Penicilliosis marneffei should be considered as a potential complication in patients with markedly impaired cell-mediated immunity who have travelled to or resided in endemic areas. Patients who have undergone therapy with purine nucleoside analogues such as fludarabine and cladribine are also at risk.     

Disseminated penicilliosis marneffei in immunocompetent patients: A report of two cases

Disseminated penicilliosis marneffei is rarely seen in immunocompetent persons. We report here two cases of disseminated penicilliosis marneffei in immunocompetent hosts. Penicillium marneffei disseminated to the brain in one patient and to the bone marrow in the other patient. Both patients received amphotericin B liposome. The cases illustrate the importance of considering penicilliosis marneffei as causes of systemic infections in immunocompetent patients.     

Usefulness of PCR and Antigen Latex Agglutination Test with Samples Obtained by Transthoracic Needle Aspiration for Diagnosis of Pneumococcal Pneumonia

In a large number of cases, the etiology of community-acquired pneumonia (CAP) is not established. Some cases are probably caused by Streptococcus pneumoniae. Transthoracic needle aspiration (TNA) culture has a limited sensitivity which might be improved by antigen detection or gene amplification techniques. We evaluated the capacity of a PCR assay and a latex agglutination test to detect S. pneumoniae in samples obtained by TNA from 95 patients with moderate-to-severe CAP. Latex agglutination and PCR had sensitivities of 52.2 and 91.3%, specificities of 88.7 and 83.3%, positive predictive values of 62.3 and 65.6%, and negative predictive values of 83.3 and 96.5%, respectively, when culture techniques were used as the “gold standard.” When we considered expanded criteria for the diagnosis of pneumococcal pneumonia as a standard for our calculations, latex agglutination and PCR had sensitivities of 53.6 and 89.7%, specificities of 93.0 and 90.0%, positive predictive values of 78.9 and 81.3%, and negative predictive values of 80.3 and 94.7%, respectively. The additional diagnosis provided by the PCR assay compared to latex agglutination was 12.2% (95% confidence interval of the difference from 0.4 to 20.1%). PCR was more sensitive than TNA culture, particularly in patients who had received prior antibiotic therapy (83.3 versus 33.3%). Although PCR is a very sensitive and specific technique, it has not proved to be cost-effective in clinical practice. Conversely, latex agglutination is a fast and simple method whose results might have significant implications for initial antibiotic therapy.     

Use of Monoclonal Antibody in Diagnosis of Candidiasis Caused by Candida albicans: Detection of Circulating Aspartyl Proteinase Antigen

To develop a serological diagnosis of invasive candidiasis based on detection of circulating secreted aspartyl proteinase (SAP) antigen of Candida albicans, three different enzyme-linked immunosorbent assays (ELISAs) were compared. The first was a standard ELISA to detect anti-SAP antibodies, and the others were an antigen capture ELISA and an inhibition ELISA to detect circulating SAP antigen with monoclonal antibody (MAb) CAP1, which is highly specific for SAP. These tests were applied to 33 serum samples retrospectively selected from 33 patients with mycologically and/or serologically proven invasive candidiasis caused by C. albicans. Serum samples from 12 patients with aspergillosis and serum samples from 13 healthy individuals were also included. The sensitivities and specificities were 69.7 and 76.0% for the standard ELISA and 93.9 and 92.0% for the antigen capture ELISA, respectively. However, these values reached 93.9 and 96.0%, respectively, for the inhibition ELISA. Serum samples from 31 of 33 patients had detectable SAP antigen, with concentrations ranging from 6.3 to 19.0 ng/ml. These results indicate that the inhibition ELISA with MAb CAP1 is effective in detection of circulating SAP antigen and that this assay may be useful for diagnosis and treatment monitoring of invasive candidiasis.     

Diagnosis of Bacteriuria and Leukocyturia by Automated Flow Cytometry Compared with Urine Culture

Urinary tract infection (UTI) is a widespread disease, and thus, the most common samples tested in diagnostic microbiology laboratories are urine samples. The “gold standard” for diagnosis is still bacterial culture, but a large proportion of samples are negative. Unnecessary culture can be reduced by an effective screening test. We evaluated the performance of a new urine cytometer, the Sysmex UF-1000i (Dasit), on 703 urine samples submitted to our laboratory for culture. We compared bacteria and leukocyte (WBC) counts performed with the Sysmex UF-1000i to CFU-per-milliliter quantification on CPS agar to assess the best cutoff values. Different cutoff values of bacteria/ml and WBC/ml were compared to give the best discrimination. On the basis of the results obtained in this study, we suggest that when the Sysmex UF-1000i analyzer is used as a screening test for UTI the cutoff values should be 65 bacteria/ml and 100 WBC/ml. Diagnostic performance in terms of sensitivity (98.2%), specificity (62.1%), negative predictive value (98.7%), positive predictive value (53.7%), and diagnostic accuracy (73.3%) were satisfactory. Screening with the Sysmex UF-1000i is acceptable for routine use. In our laboratory, we have reduced the number of bacterial cultures by 43%, speeded up their reporting, and decreased the inappropriate use of antibiotics.The urinary tract is the most common site of infection, and urine culture is the standard diagnostic test for urinary tract infection (UTI). The growing need to enhance the performance of urine culture combined with the need to free up resources by rejecting negative samples quickly and economically has drawn attention to solutions that can be used as screening tests to reduce the number of unnecessary culture tests. In this study, we sought to evaluate and optimize the performance of the Sysmex UF-1000i (Dasit) on 703 urine samples submitted at the same time to bacterial culture analysis.     

Interpretations of Antibody Responses to Salmonella enterica Serotype Enteritidis gm Flagellin in Poultry Flocks Are Enhanced by a Kinetics-Based Enzyme-Linked Immunosorbent Assay

Many regulatory and diagnostic programs for the detection of Salmonella enterica serotype Enteritidis infection in commercial poultry flocks have relied on rapid Pullorum agglutination tests to screen birds because of the shared antigens of S. enterica Enteritidis and S. enterica Pullorum and Gallinarum; however, the use of the enzyme-linked immunosorbent assay (ELISA) format affords better analytical sensitivity than crude agglutination tests. In this study, we adapted our earlier conventional indirect ELISA, using gm flagellin as the antigen, to a kinetics-based, computer-controlled ELISA (KELA). The KELA was used to screen for flagellin antibody from three commercial flocks: (i) a large flock involved in a U.S. Department of Agriculture trace back from a human S. enterica Enteritidis foodborne outbreak (n = 3,209), (ii) a flock infected with the endemic S. enterica Enteritidis serotype but which also had multiple other salmonella serotypes (n = 65), and (iii) an S. enterica Pullorum-infected flock (n = 12). The first flock (S. enterica Enteritidis prevalence of 2.45% based on culture) provided a field test of the KELA and allowed the calculation of diagnostic sensitivity (D-Sn) and diagnostic specificity (D-Sp). With a cutoff of 10 (used for screening flocks [i.e., high sensitivity]), the KELA has a D-Sn of 95.2% and a D-Sp of 18.5%; with a cutoff of 140 (used in confirmatory flock testing [i.e., high specificity]), the KELA has a D-Sn of 28.0% and a D-Sp of 99.1%. We found that with a cutoff of 60 (D-Sn = 63.1%; D-Sp = 91.6%), we could eliminate reactions in the KELA caused by other non-S. enterica Enteritidis salmonellae. The KELA was also compared to two commercial rapid Pullorum tests, the Solvay (D-Sn = 94.9%; D-Sp = 55.5%) and the Vineland (D-Sn = 62.0%; D-Sp = 75.3%).     

Detection of Lassa Virus Antinucleoprotein Immunoglobulin G (IgG) and IgM Antibodies by a Simple Recombinant Immunoblot Assay for Field Use

The nucleoprotein of Lassa virus, strain Josiah, was expressed in Escherichia coli as an N-terminally truncated, histidine-tagged recombinant protein. Following affinity purification the protein was completely denatured and spotted onto nitrocellulose membrane. A total of 1 μg of protein was applied for detection of Lassa virus antibodies (LVA) in a simple immunoblot assay. Specific anti-Lassa immunoglobulin M (IgM) antibodies could be detected by increasing the amount of protein to 5 μg. A panel of 913 serum specimens from regions in which Lassa virus was endemic and from regions in which Lassa virus was not endemic was used for evaluating the sensitivity and specificity of the LVA immunoblot in comparison to those of an indirect immunofluorescence (IIF) assay. The sera originated from field studies conducted in the Republic of Guinea (570 serum samples) and Liberia (99 serum samples), from inpatients of the clinical department of the Bernhard-Nocht-Institute, Hamburg, Germany (94 serum samples), and from healthy German blood donors (150 serum samples). In comparison to the IIF assay the LVA immunoblot assay had a specificity of 90.0 to 99.3%, depending on the origin of the specimens. The sensitivity was found to be highest for the Guinean samples (90.7%) and was lower for the Liberian samples (75%). Acute Lassa fever was diagnosed by PCR in 12 of 59 (20.3%) patients with fever of unknown origin (FUO) from the Republic of Guinea. On admission to the hospital, nine Lassa fever patients (75%) were reactive by the IgM immunoblot assay. One of the patients was infected with a new Lassa variant, which showed 10.4% variation on the amino acid level in comparison to the prototype strain of Lassa virus, Josiah. Seven PCR-negative patients were reactive by immunoblotting. The positive and negative predictive values of a single IgM immunoblot result for acute, PCR-confirmed Lassa fever were therefore 53.6 and 93.0%, respectively. Because of its high negative predictive value, a single IgM immunoblot result will be valuable for excluding acute Lassa fever for cases of FUO in areas where Lassa fever is endemic.     

Detection of circulating Aspergillus fumigatus DNA by real-time PCR assay of large serum volumes improves early diagnosis of invasive aspergillosis in high-risk adult patients under hematologic surveillance

Detection of galactomannan antigen (GMA) in serum is the standard assay for the diagnosis of invasive aspergillosis (IA) in high-risk patients with hematological disorders. Detection of Aspergillus DNA in serum has been proposed, but its sensitivity is lower than that of GMA when small serum volumes (SSV) are used. In this study, we investigated whether extraction of DNA from large serum volumes (LSV) improves diagnostic yield. In a 13-month prospective study, we compared the performances of twice-weekly screening of serum for GMA by an enzyme immunoassay and weekly screening for Aspergillus fumigatus DNA by a real-time PCR (RT-PCR) assay of 1.0 ml (LSV) or 100 μl (SSV) of serum. We included 124 patients (138 treatment episodes), with 17 episodes of EORTC (European Organization for Research and Treatment of Cancer)/MSG (Mycoses Study Group)-documented IA. In all, 1,870 samples were screened for GMA. The sensitivity (Se), specificity (Sp), and positive and negative predictive values (PPV and NPV, respectively) of GMA for IA were 88.2%, 95.8%, 75%, and 98.3%, respectively. We screened 938 samples for Aspergillus DNA by using LSV; 404 of these samples were also tested with SSV. The Se, Sp, PPV, and NPV of RT-PCR were 100%, 96.7%, 81%, and 100%, respectively, with LSV and 76.5%, 96.7%, 81.3%, and 95.6%, respectively, with SSV. DNA detection gave a positive result when performed on LSV in two cases of IA where the GMA assay result remained negative. Furthermore, in four IA cases, DNA was detected earlier than GMA. The use of LSV for extraction improved the performance of the RT-PCR, which appears highly sensitive and specific for the early diagnosis of IA in high-risk patients with hematological disorders.