Detection Of Haemophilus Influenzae Type Research Articles

DNA aptamers for the detection of Haemophilus influenzae type b by cell SELEX.

Haemophilus influenzae type b (Hib) causes acute bacterial meningitis (ABM) in children, with a mortality rate of about 3-6% of the affected patients. ABM can lead to death during a period of hours to several days and, hence, rapid and early detection of the infection is crucial. Aptamers, the short single-stranded DNA or RNA with high affinity to target molecules, are selected by a high-flux screening technique known as in vitro screening and systematic evolution of ligands by exponential enrichment technology (SELEX). In this study, whole-cell SELEX was applied for the selection of target-specific aptamers with high affinity to Hib. ssDNA aptamers prepared by lambda exonuclease were incubated with the target cells (Hib). The aptameric binding rate to Hib was characterized for binding affinity after seven SELEX rounds by flow cytometry. The aptamers with higher binding affinity were cloned. Four of 68 aptamer clones were selected for sequencing. The dissociation constant (Kd) of the high-affinity aptamer clones 45 and 63 were 47.10 and 28.46pM, respectively. These aptamers did not bind to other bacterial species, including the seven meningitis-causing bacteria. They showed distinct affinity to various H. influenzae strains only. These aptamers showed the highest affinity to Hib and the lowest affinity to H. influenzae type c and to other meningitis-causing bacteria. Clone 63 could detect Hib in patients' cerebrospinal fluid (CSF) samples at 60colony-forming units (CFU)/mL. The results indicate applicability of the aptamers for rapid and early detection of infections brought about by Hib.

Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

Loop-Mediated Isothermal Amplification Assay for Detection of Haemophilus influenzae Type b in Cerebrospinal Fluid

Haemophilus influenzae type b (Hib) is one of the leading causes of meningitis in developing countries. To establish and evaluate a novel loop-mediated isothermal amplification (LAMP) assay for Hib, we designed a LAMP primer set targeting the Hib-specific capsulation locus. LAMP detected 10 copies of purified DNA in a 60-min reaction. This indicated that the detection limit of LAMP was >100-fold lower than the detection limits of both a PCR for the detection of bexA and a nested PCR for Hib (Hib PCR). No H. influenzae, other than Hib or control bacteria, was detected. Linear determination ranged from 10 to 1,000,000 microorganisms per reaction mixture using real-time turbidimetry. We evaluated the Hib LAMP assay using a set of 52 randomly selected cerebrospinal fluid (CSF) specimens obtained from children with suspected meningitis. For comparison, the CSF specimens were tested using a conventional Hib PCR assay. Hib was detected in 30 samples using LAMP and in 22 samples using the Hib PCR assay. The Hib PCR showed a clinical sensitivity of 73.3% and a clinical specificity of 100% relative to the Hib LAMP assay. These results suggest that further development and evaluation of the Hib LAMP will enhance the global diagnostic capability for Hib detection.

Detection of Haemophilus influenzae type B DNA in a murine pneumonia model by in situ PCR

This study estimated the value of in situ PCR (ISPCR) in the detection of Haemophilus influenzae type b (Hib) DNA in paraffin-embedded lung tissues of a murine pneumonia model. ICR mice were infected with Hib solution intranasally. In study group A (n=20), physiological changes and the number of deaths were recorded for 7 consecutive days after infection. In study group B (n=10), blood samples and lung tissues were obtained from the infected mice on the brink of death. In both groups, portions of the lung tissue were cultured for Hib, while other portions were submitted for histopathological studies. Conventional PCR, PCR followed by Southern blotting and ISPCR were performed to detect Hib in paraffin-embedded lung tissues. In control group A, six mice were inoculated intranasally with the same concentration of heat-inactivated Hib solution. In control group B, six healthy mice served as a blank control. Both control groups were managed using the same methods as those used in the study groups. The white blood cell count of the mice in the study group increased (F=3.295, P<0.01), with a high neutrophil count (F=0.127, P<0.05). In the histopathological study, various stages of pneumonia were found in the lung tissues of the infected mice examined by microscope; 80% of the mice had moderate or severe pneumonia. Cultures of lung tissues in the study groups were all positive for Hib, while no bacteria were found in the control groups. Hib was detected in only 4 of 30 samples (13.3%) of the study groups using conventional PCR, but in all 30 samples (100%) using both Southern blotting and ISPCR. All three methods did not detect Hib in the control groups. Because of its sensitivity and specificity and its ability to locate the micro-organism, ISPCR can be considered suitable for the detection of Hib in paraffin-embedded lung tissues.

Specific detection of Haemophilus influenzae type b lipooligosaccharide by immunoassay

Three monoclonal antibody (MAb)-based immunoassays were developed for specific detection of Haemophilus influenzae type b (Hib) lipooligosaccharide (LOS). (i) Hib LOS was captured onto microtiter plates by polyclonal Hib-directed antibodies and detected with MAbs to the oligosaccharide component of Hib LOS in an enzyme-linked immunosorbent assay, (ii) The high affinity of polymyxin B for lipid A was used to bind Hib LOS to microtiter wells, and the oligosaccharide-specific MAbs were used as the detection system in the polymyxin B-MAb assay. (iii) Hib LOS solubilized in detergent was captured by MAbs, and the immobilized LOS was detected with a chromogenic Limulus amebocyte lysate method in the immunolimulus assay. Endotoxin concentrations were measured in in vitro samples and cerebrospinal fluid samples from rabbits with experimental Hib meningitis. The results were compared with those obtained with the standard chromogenic Limulus amebocyte lysate assay. There were significant correlations between the results of all four assays. These new immunoassays provide methods for specific detection of Hib LOS in laboratory fluids and in research involving quantification of Hib endotoxin in experimental animal models.

Rapid diagnosis of acute bacterial meningitis by coagglutination and latex agglutination

Early diagnosis and initiation of appropriate antimicrobial therapy is essential for the reduction of mortality and morbidity in acute pyogenic meningitis. The rapid diagnosis of meningitis by the detection of bacterial antigens in the cerebrospinal fluid (CSF) is now possible with the availability of commercially prepared kits. One hundred and eighty-six CSF samples were cultured for bacteria and examined for antigens of common organisms causing meningitis by a coagglutination technique (COA) and a latex agglutination test (LA). The COA had a concordance rate of 95·7% with culture while that of LA was 92·9%. The detection of Haemophilus influenzae type b antigens by both kits were in complete agreement with culture results ( 15 15 ). With Streptococcus pneumoniae antigens the COA was slightly more sensitive than the LA. With the Streptococcus group B antigen, however, both the tests proved to be oversensitive thus giving rise to false positives. Both kits failed to detect Neisseria meningitidis serogroup B exoantigens in two culture positive cases. Thus, while rapid diagnosis of acute bacterial meningitis by the detection of antigens in the CSF is extremely useful, negative results do not exclude the possibility of meningitis and positive results should be interpreted with the clinical picture and other laboratory parameters.

Specific detection of Haemophilus influenzae type b lipooligosaccharide by a polymyxin B monoclonal antibody assay

A monoclonal antibody (MAb)-based enzyme immunoassay was developed for detection of Haemophilus influenzae type b (Hib) lipooligosaccharides (LOS). The high affinity of polymyxin B for lipid A was used to bind the Hib LOS to microtiter wells. The immobilized LOS was detected with MAbs directed against the oligosaccharide component of Hib endotoxin. Hib LOS concentrations were measured in in vitro samples and in cerebrospinal fluid (CSF) samples obtained from rabbits with experimental Hib meningitis. The sensitivity of the assay was 1 ng LOS/ml sample and the results obtained with this assay correlated significantly with those obtained with the standard Limulus amebocyte lysate assay. This new assay provides a method for specific detection of Hib LOS in CSF samples and in aqueous laboratory fluids. This general methodology should also be useful for experimental research involving specific LPS/LOS molecules.

Preparation of urine samples for use in commercial latex agglutination tests for bacterial antigens.

The use of latex agglutination (LA) tests for bacterial antigen detection in urine specimens is hindered by troublesome reactions such as nonspecific agglutination. Therefore, procedures such as boiling or membrane filtration of urine specimens are often used before LA testing. We discovered that the composition of the membrane filter used in filtration has a marked effect on the performance of an LA test used for detection of Haemophilus influenzae type b antigen. False-positive LA reactivity was common in urine specimens from pediatric patients that were processed by membrane filtration through certain filters; furthermore, such reactivity also occurred in LA tests for antigens other than those of H. influenzae. A protein present in urine at low concentrations appeared to be responsible for these phenomena.

Detection of Haemophilus influenzae type b antigenuria by Bactigen and Phadebact kits.

Two commercially available reagents, latex particles (Bactigen) and Staphylococcus aureus suspensions (Phadebact), were compared for the detection of the capsular polyribitol phosphate antigen of Haemophilus influenzae type b in 18 pediatric patients with proven infections due to H. influenzae type b. Whereas both tests nearly equally detected the antigen in the first urine specimens from the patients, the latex test remained positive significantly longer than did the Phadebact test for serial urine specimens. We conclude that the Bactigen test is slightly more sensitive than the Phadebact test for detecting urinary H. influenzae type b antigen.

Comparison of coagglutination, latex agglutination, and enzyme-linked immunosorbent assay for detection of Haemophilus influenzae type B infection

Two commercially available kits were compared to an enzyme-linked immunosorbent assay (ELISA) for detection of Haemophilus influenzae type b antigen polyribophosphate in clinical specimens with infections proved by culture. Methods employed by the kits were latex agglutination (LA) and staphylococcal coagglutination (COA). The COA kit detects H. influenzae type b and types a and c-f with a polyvalent antiserum, whereas the ELISA assay and the LA kit detect only type b. A total of 139 specimens (41 spinal fluid, 35 urine, 25 serum, and 38 sputum) were tested. All spinal fluid samples positive by culture were positive by all three procedures. Of urine specimens from patients with a variety of H. influenzae type b infections, 13 of 15 were positive by ELISA, 8 of 15 by COA, and 8 of 14 by LA. Of serum samples collected from the same patients at various times during their illness, 8 of 15 were positive by ELISA, 6 of 15 by COA, and 10 of 15 by LA. Of sputum samples positive by culture for H. influenzae type b, 14 of 17 were positive by ELISA, 9 of 17 by COA, and 4 of 16 by LA. The ability to detect additional serotypes of H. influenzae was shown by the COA kit, which detected H. influenzae type a in spinal fluid from a patient with type a meningitis proved by culture.

Evaluation of Bactogen and Phadebact for detection of Haemophilus influenzae type b antigen in cerebrospinal fluid.

Latex agglutination and coagglutination tests are commercially available as Bactogen and the Phadebact Haemophilus Test, respectively. We evaluated both for the detection of Haemophilus influenzae type b in cerebrospinal fluids. Both tests were positive in all of 51 culture-positive cases of meningitis caused by H. influenzae. Both were more sensitive than counterimmunoelectrophoresis. Antigen was also detected by Bactogen in seven of seven additional cerebrospinal fluid specimens (compared with four of seven by Phadebact) after 1 to 15 days of antimicrobial therapy. The cerebrospinal fluid of infants with meningitis owing to other common causative agents did not react with Bactogen or Phadebact. However, the cerebrospinal fluid of one patient with overwhelming infection owing to Proteus mirabilis reacted positively with Bactogen. Cost analysis revealed that Phadebact was less expensive to perform than Bactogen.

Clinical studies of a new latex particle agglutination test for detection of Haemophilus influenzae type b polyribose phosphate antigen in serum, cerebrospinal fluid, and urine.

A new latex particle agglutination test for direct detection of Haemophilus influenzae type b polyribose phosphate antigen in serum, cerebrospinal fluid, or urine was evaluated from studies at four clinical centers. Although use of a serum buffer significantly reduced inconclusive agglutination of the latex particles, the retesting of serum samples, after heat inactivation and dilution, resolved all serum samples, with one exception, as reactive or nonreactive for the presence of the polyribose phosphate antigen. A clinical accuracy of 100% was obtained for the latex particle agglutination method in respect to its capability for detection of polyribose phosphate antigen in all patients with confirmed infection by H. influenzae type b.

Rapid detection of Haemophilus influenzae type b in cerebrospinal fluid by commercial coagglutination and latex agglutination kits.

The ability of two commercial kits to detect Haemophilus influenzae type b antigen in cerebrospinal fluid was evaluated. Results obtained by Bactogen, a latex agglutination test, and Phadebact, a coagglutination test, were compared to counterimmunoelectrophoresis, Gram stain and culture results. One hundred and seven specimens of cerebrospinal fluid were tested. Thirty were found to contain bacteria, 20 of which were Haemophilus influenzae type b. All 20 were positive by Bactogen and Phadebact testing, 19 were culture positive, 18 were positive by counterimmunoelectrophoresis and 15 had gram-negative bacilli seen on Gram stain. The culture negative specimen contained microscopically visible gram-negative bacilli and was from a patient on antimicrobial therapy who was previously Haemophilus influenzae type b culture positive. No false positives from other genera or the 77 culture negative specimens occurred with Phadebact, Bactogen or counterimmunoelectrophoresis.

Haemophilus influenzae type b antigenuria in children.

Detection of Haemophilus influenzae type b (HIb) antigenuria by latex agglutination has been shown to be sensitive, specific, and rapid. In children, antigenuria persisted for a mean duration of 10 days and a maximum of 18 days. Antigenuria was demonstrated in 25 of 30 patients with HIb infection but not in 62 with other types of infection. In five children, antigenuria confirmed the diagnosis in the absence of bacteriological confirmation. In five other children, antigenuria was not found, but in this group the antigen was detected in another body fluid or HIb was recovered.