Laboratory diagnosis of Mycoplasma pneumoniae infection. 1. Direct detection of antigen in respiratory exudates by enzyme immunoassay

Direct and indirect antigen capture enzyme immunoassays (Ag-EIA) have been developed for the detection of Mycoplasma pneumoniae in nasopharyngeal aspirates or sputum from respiratory infection. The sensitivity of the two Ag-EIA were similar, but the indirect method using polyclonal rabbit and guinea-pig antisera was more convenient. The Ag-EIA had a detection limit of 10(4-4.5) colony-forming units/ml of sample. It was specific for M. pneumoniae and gave a low level response with M. genitalium. There were no cross-reactions with 10 other species of mycoplasmas. Tests with a wide range of bacteria and chlamydia group antigen, representing agents sometimes found in the respiratory tract, were also negative. At the current level of development, the Ag-EIA detected about 90% of specimens that were also positive for culture; 43% of specimens from culture-negative--seropositive patients gave a positive result. The overall pattern of results indicated that while antigen detection is a quick and effective substitute for the slow culture method, serological examination for specific IgM antibody is also necessary to give a complete diagnostic coverage.     

Laboratory diagnosis of Mycoplasma pneumoniae infection. 4. Antigen capture and PCR-gene amplification for detection of the Mycoplasma: problems of clinical correlation.

Direct detection assays for Mycoplasma pneumoniae were established by PCR amplification of short sequences within the foot protein/adhesin (P1) gene and the 16S ribosomal RNA gene. Specificity and sensitivity was excellent, no hybridization was observed with M. genitalium and other human Mycoplasma species. In nose and throat washings from subjects with respiratory infection a pattern of high counts (c.f.u./ml) of M. pneumoniae (deduced from the amount of amplified PCR product), and a positive antigen capture assay, was found in 83% of subjects with serological evidence of current infection with M. pneumoniae. A small proportion of subjects with serological patterns suggesting infection in the more distant past had positive PCR assays. This was considered to represent either persistence of the organism from a previous infection or perhaps transient carriage during a reinfection, without substantial change in antibody response. PCR-based assay of M. pneumoniae offers a powerful, rapid, and sensitive substitute for culture of the mycoplasma. Antigen capture, while less sensitive than PCR, offers the advantage that it is more often positive with samples from current infection and requires less stringent laboratory organization to contain false positive results. We conclude however that the laboratory diagnosis of a chosen clinical episode should not rest on the PCR or Ag-EIA assays alone, but must also include antibody assays to confirm whether infection is current or represents persistence from past exposure.     

Laboratory diagnosis of Mycoplasma pneumoniae infection. 3. Detection of IgM antibodies to M. pneumoniae by a modified indirect haemagglutination test

The indirect haemagglutination (IHA) test was compared with the complement-fixation (CF) test for the measurement of antibodies to Mycoplasma pneumoniae. A modification of the IHA was used to measure M. pneumoniae IgM antibodies. Sera were obtained from various groups of patients who were either culture or antigen positive for M. pneumoniae in nasopharyngeal aspirates or who had fourfold or greater increase in CF antibody or a titre greater than or equal to 320. The results of these comparisons showed that the modified IHA test was specific and more sensitive (89% as opposed to 64%) than the CF test. The modified IHA test for the detection of IgM antibody was highly effective in the recognition of recent or current infection with the mycoplasma. It was also of equal sensitivity to an indirect enzyme immunoassay for the detection of IgM antibodies to M. pneumoniae.     

A micro-capture ELISA for detecting Mycoplasma pneumoniae IgM: comparison with indirect immunofluorescence and indirect ELISA.

A mu-capture ELISA was developed for detecting Mycoplasma pneumoniae-specific IgM, and compared with an indirect immunofluorescent antibody (IFA) technique and an indirect ELISA. mu-capture ELISA and IFA compared well and were found to be the most sensitive assays. The IFA test can be completed in 2 h whilst the results of the mu-capture ELISA can be available in 24 h. Both tests are amenable to routine diagnostic use and have similar sensitivity. Indirect ELISA was found to be less sensitive and less specific, giving high assay values with several sera having undetectable M. pneumoniae CF antibody or CF antibody in low titre. Serum samples obtained from 11 patients at various times after M. pneumoniae infection showed maximum antibody levels within the first month by all assays, with a gradual fall in amount of IgM with time when assayed by mu-capture ELISA, a more gradual decline by IFA and hardly any decline with indirect ELISA. It was concluded that the indirect ELISA is unsuitable for the investigation of possible M. pneumoniae infection because the sustained high assay values with serum samples taken many months after infection, make interpretation of the test results very difficult.     

Jejunal flora of patients with megaoesophagus secondary to Chagas disease

The jejunal flora of 15 patients with megaoesophagus secondary to Chagas disease was studied and compared with that of 15 control individuals. In addition to the serological reactions for Chagas disease (immunofluorescence and complement fixation reaction), all subjects were submitted to endoscopy and X-ray of the oesophagus, gastric secretory study and investigation of the jejunal flora. The mean bacterial counts (log10) of Chagas disease patients (4.14 +/- 2.15 c.f.u./ml) was significantly higher than those of the control group (2.83 +/- 1.34 c.f.u./ml). Aerobic bacteria were isolated from 14 Chagasic patients (maximum count 10(10) c.f.u./ml) and 7 controls (maximum count 10(5) c.f.u./ml). Anaerobes were isolated from 7 patients (maximum count 10(7) c.f.u./ml) and 1 control (10 c.f.u./ml). Controls and Chagas disease patients differed significantly in the maximum acid output, but there was no statistically significant relation between bacterial counts and maximum output.     

Bacterial contamination of floors and other surfaces in operating rooms: a five-year survey.

Bacterial contamination of floors and other surfaces in the operating suite has been investigated by contact impression plates during the past five years. Colony counts of the floors of operating rooms, cleaned with disinfectant, were 3.3 c.f.u./10 cm2; on the floors of semi-clean and dirty areas, cleaned with detergent, colony counts were 44.8 and 71.4 c.f.u./10 cm2 respectively. The highest colony counts of 487.4 c.f.u./10 cm2 were found in the dressing rooms, the floors of which were covered with carpets, cleaned with a vacuum cleaner. Mean bacterial numbers on surfaces of various equipment in operating rooms, cleaned with disinfectant, were 2.8 c.f.u./10 cm2. Bacterial numbers on surfaces decreased markedly from 253.2 to 11.9 c.f.u./10 cm2 following the use of disinfectant. Bacterial species found from various surfaces were mainly coagulase-negative staphylococci, derived from human beings. In the light of these findings the regular use of disinfectant for cleaning of the floors and other surfaces in operating rooms is advisable.     

Synergy of four macrolide antibiotics with chloroquine against chloroquine-resistant Plasmodium falciparum in vitro

The antimalarial activity of four macrolide antibiotics was investigated against the multidrug resistant K1 strain of Plasmodium falciparum in vitro. ID50 (50% inhibitory concentration) values for erythromycin, spiramycin, tylosin tartrate and oleandomycin phosphate in 48-hour assays were 1.6 X 10(-4)M, 2.5 X 10(-5)M, 1.2 X 10(-5)M and 9 X 10(-6)M respectively, and in 96 hour assays were 10(-5)M, 2.6 X 10(-6)M, 2.6 X 10(-6) and 3 X 10(-6)M, respectively. Comparable values were obtained in assays in which drug effect was quantified from either parasite counts or 14C isoleucine incorporation. Each of the four macrolides displayed synergy with chloroquine at the IC90 (90% inhibitory concentration) level, but at the IC50 level synergy was either less pronounced or absent. For each combination this difference in the degree of synergy was significant at the 95% level of confidence. In replicate assays in which 3H hypoxanthine was the marker of drug effect, synergy between chloroquine and either erythromycin or spiramycin could not be detected.     

Rubella antibody measured by radial haemolysis. Characteristics and performance of a simple screening method for use in diagnostic laboratories.

A simple method for preparing radial haemolysis gels for rubella antibody screening is described. In use it gave clear zones of haemolysis when a standard serum was tested at dilutions down to 5.6 i.u./ml rubella antibody. In five laboratories 8404 sera were screened by the method and the results were read by comparing zones of haemolysis with that of a standard serum diluted to contain 15 i.u/ml antibody. A zone greater than or equal to 15 i.u./ml, indicating immunity, was given by 7433 (88.4%) of the sera. No zone indicating susceptibility was seen with 748 (8.9%) sera. Small zones, less than 15 i.u./ml standard, were given by 189 (2.2%) sera, and in only 34 cases (0.4%) did non-specific haemolysis interfere with the test readings. Further testing of the radial haemolysis interfere with the test readings. Further testing of the radial haemolysis negative and low positive sera by the haemagglutination inhibition test gave rise to some discrepant results which are discussed.     

A comparison of three vaccines against respiratory syncytial virus in calves.

An inactivated vaccine against respiratory syncytial virus (RSV) was compared with two live vaccines. The inactivated (GC) vaccine consisted of glutaraldehyde-fixed bovine nasal mucosa cells persistently infected with RSV and emulsified with oil adjuvant. The live vaccines were a modified virus (MV) derived from a bovine strain of RSV and a temperature-sensitive mutant (ts-1) derived from a human strain. The GC vaccine was inoculated subcutaneously into 12 calves and the live vaccines intramuscularly into eight calves each. Nine unvaccinated calves acted as controls. The vaccines were administered in two doses 3 weeks apart and all calves were challenged intranasally with 2 X 10(7) p.f.u. of bovine RSV 3 weeks after the second dose. At the time of challenge calves given GC, MV and ts-1 vaccines had mean serum neutralizing antibody titres of 25, 19 and 2 respectively; mean titres of IgG1 antibody by radioimmunoassay were log10 4.5, 1.3 and 2.6 respectively and mean zone areas by single radial haemolysis (SRH) were 107, 27 and 36 mm2 respectively. Eleven of 12 calves given GC vaccine were completely protected against challenge but all control animals and those given the two live vaccines were infected. The mean peak titre of virus in nasal swabs of control calves was 3.0 log10 p.f.u./ml and the mean duration of virus shedding was 6.8 days. Both these parameters were significantly reduced in animals given MV and ts-1 vaccines: mean peak titres were 2.1 and 2.4 log10 p.f.u./ml and mean duration of shedding was 3.4 and 3.3 days respectively. Thus, protection correlated better with RSV antibody detected by radioimmunoassay and SRH than with neutralizing antibody. These results are discussed in relation to the possible mechanism by which protection was mediated.     

ELISA法在检测小儿肺炎支原体感染中的应用

目的 探讨ELISA法在检测小儿肺炎支原体感染中的应用.方法 选取2010年6月- 2011年9月126例小儿肺炎支原体感染患儿进行研究,将患儿分为ELISA检测组和荧光定量PCR法检测组,各63例,统计各组患者检测的阳性率及肺炎患病类型.结果 ELISA检测组有15例阳性,阳性率为23.81％,支气管哮喘2例占13.33％,支气管肺炎6例占40.00％,急性支气管炎4例占26.67％,上呼吸道感染2例占13.33％,其他1例占6.67％;荧光定量PCR法检测组有26例阳性,阳性率为41.26％,支气管哮喘4例占15.38％,支气管肺炎11例占42.31％,急性支气管炎6例占23.08％,上呼吸道感染3例占11.57％,其他2例占7.69％.结论 使用ELISA法在检测小儿肺炎支原体感染中的效果好,建议在检测小儿肺炎支原体感染中广泛使用.     

Detection of photosynthetic herbicides: Algal growth inhibition test vs. electrochemical photosystem II biosensor

We compared a novel PSII-biosensor assay with a standard algal growth inhibition test for detection of photosynthetic herbicides—diuron, atrazine and isoproturon in liquid samples. To evaluate the convenience and sensitivity, values of the parameters EC50 and LOD and the duration of assays were compared. The biosensor assay was made with an electrochemical biosensor toxicity analyser with immobilised Photosystem II (PSII) complex. Using the PSII-biosensor assay, higher sensitivity (LOD) to herbicides (10⁻⁸-10⁻⁹ M) was achieved as compared to standard algal growth inhibition tests (about 10⁻⁷ M). The results of both assays showed a good correlation as concerns their EC50 values while the interval of detectable concentrations is about twice wider for PSII-biosensor. A proposed measurement protocol includes the reference standard of phytotoxicity (RSP). The main advantage of the PSII-biosensor assay is that it can be completed in about 1 h and is by 1-2 orders more sensitive than standard algal growth inhibition test, which takes 72 h.     

Interaction of L. pneumophilia and a free living amoeba (Acanthamoeba palestinensis)

Co-cultivation of Legionella pneumophila serogroup I and Acanthamoeba palestinensis in Neff's medium at 35 degrees C resulted in the intracellular multiplication of the bacteria as demonstrated by electron microscopy and immunofluorescence. In the closed experimental system used, the number of legionellae rose from 10(7) colony forming units (c.f.u.)/ml initially to a maximum of 10(10) c.f.u./ml on day 5. Legionellae were seen in expelled phagosomes, in some amoebae filling the cytoplasm and in others in which the process of encystment appeared to have commenced. At 20 degrees C the acanthamoebae phagocytosed and digested the legionellae. The bacteria disappeared from the co-cultivation flask by day 2 but reappeared in low numbers (10(2) c.f.u./ml) by day 6 suggesting that even at this temperature some intra-amoebal multiplication occurred.     

Interaction of L. pneumophilia and a free living amoeba (Acanthamoeba palestinensis).

Co-cultivation of Legionella pneumophila serogroup I and Acanthamoeba palestinensis in Neff''s medium at 35 degrees C resulted in the intracellular multiplication of the bacteria as demonstrated by electron microscopy and immunofluorescence. In the closed experimental system used, the number of legionellae rose from 10(7) colony forming units (c.f.u.)/ml initially to a maximum of 10(10) c.f.u./ml on day 5. Legionellae were seen in expelled phagosomes, in some amoebae filling the cytoplasm and in others in which the process of encystment appeared to have commenced. At 20 degrees C the acanthamoebae phagocytosed and digested the legionellae. The bacteria disappeared from the co-cultivation flask by day 2 but reappeared in low numbers (10(2) c.f.u./ml) by day 6 suggesting that even at this temperature some intra-amoebal multiplication occurred.     

Human bocavirus infection, People's Republic of China

A newly identified parvovirus, human bocavirus (HBoV), was found in 21 (8.3%) of 252 nasopharyngeal aspirates from hospitalized children with lower respiratory tract infection in Hunan Province, People's Republic of China. Viral loads were 10(4) to 10(10) copies/mL. Phylogenetic analysis of the VP1 gene showed a single genetic lineage of HBoV worldwide.     

Listeria species in domestic environments.

Using a direct isolation method Listeria spp. were detected in 101 (47.4%) of 213 houses investigated. L. monocytogenes was present in 45 houses (21.1%). Listeria spp. occurred at all sampling sites. Dish-cloths (37%) and surface samples round the drain in the bathroom (27.2%) were most frequently contaminated. Highest numbers (c. 10(4) c.f.u./object) were found in dish-cloths and washing-up brushes. Lower levels (up to 10(3) c.f.u./object) were obtained from kitchen sinks, refrigerator vegetable compartment samples and tooth brushes. In total, 132 isolations of Listeria spp. were made from 871 samples. L. innocua (53%) and L. monocytogenes (41%) were the predominant species in the positive samples. Other Listeria spp. were found in only 6% of the positive samples.     

Evaluation of an acute point-of-care system screening for respiratory syncytial virus infection

There continues to be a significant risk of children contracting hospital-acquired infections caused by respiratory syncytial virus (RSV). In order to provide 24 h screening, we examined a point-of-care system (near-patient testing) for use by non-laboratory healthcare workers (HCWs) in a short stay unit adjoining the accident and emergency department of a large paediatric hospital. Three studies were conducted over consecutive winter epidemics, in which 2193 nasopharyngeal aspirates were obtained from children < 2 years old. An average of 23 trained HCWs tested aspirates with the Abbott TESTPACK(R) RSV assay. Material was sent to the virology laboratory for examination for RSV and other respiratory viruses by direct immunofluorescence. The mean performance characteristics of near patient testing were sensitivity 90%, specificity 92%, positive predictive value 92% and negative predictive value 92%. This was acceptable for clinical purposes. The near-patient testing provided a rapid answer and ensured that infants could be segregated according to infection status. Early antiviral treatment could be commenced and needless antibiotics avoided. During the study the hospital-acquired infection rate was the lowest recorded, although this may have been influenced by national trends and lower rates of inpatient care for infants with bronchiolitis.     

非典型病原体引起下呼吸道感染的调查

目的：了解肺炎支原体和肺炎衣原体在下呼吸道感染率。为经验用药提供依据。方法：双盲法从15所医院收集128位临床诊断疑由肺炎支原体或肺炎衣原体，引起的下呼吸道感染患者在不同病程收集血清标本335份，用固相酶联免疫吸附检测肺炎支原体IgG和IgM，肺炎衣原体IgG和IgM。结果：近期感染：单一肺炎支原体感染患者占16．4％，单一肺炎衣原体感染占17．2％，混合感染患者占17．2％，近期总感染率：50．8％；近期＋既往总感染率：80．5％，其中；肺炎支原体感染患者占49．2％，肺炎衣原体感染占52．3％，成人和儿童的近期感染率分别为41．7％和78．1％，结论：肺炎支原体和肺炎衣原体是社区获得下呼吸道感染的主要病原体。     

Response of adult human volunteers to oral administration of bovine and bovine/human reassortant rotaviruses

Small groups of adult volunteers, in sequence, were inoculated orally with inactivated purified bovine rotavirus of strain NCDV, with live NCDV purified or unpurified and with two different NCDV X human rotavirus reassortant viruses. One of five volunteers given 200 micrograms of ultravioletinactivated NCDV developed a virus-neutralizing (VN) and a binding antibody response detected by enzyme-linked immunosorbent assay (ELISA). Four of 10 volunteers given from 1 X 10(6) to 1 X 10(8) p.f.u. of live NCDV developed VN antibody, but nine of 10 responded when ELISA, HAI and radioimmuno-precipitation tests for serum antibody were also considered. Two different NCDV X human serotype 1 Wa strain virus reassortants, each containing Wa gene segment 9 and the serotype 1 neutralization phenotype, were administered orally in doses up to 10(6) p.f.u. The reassortants were relatively ineffective in eliciting a serum antibody response at the dosage level employed.     

多种呼吸道病原微生物快速筛查技术的建立

目的解决临床急性呼吸道感染病原微生物快速筛查诊断的难题,建立一种能同时快速检测多种病原体的有效方法。方法采用多重PCR与Luminex xMAP多重分析技术平台相结合,以荧光微球为检测载体,建立快速、多重检测DNA或RNA技术平台。结果经14种(18个亚型)常见呼吸道病原微生物标准株检测,在反应体系中多重PCR产物只与相应的病原菌检测微球杂交,无非特异性反应;通过138例临床标本检测验证,肺炎患者支气管肺泡灌洗液标本中,细菌检出率占总病原微生物的37.68%,其中结核分枝杆菌占18.84%;病毒检出率占总病原微生物的44.93%,支原体、衣原体占17.39%;患者支气管肺泡灌洗液和鼻咽拭子标本中流感病毒A型的检出率分别为18.75%和26.92%。结论多重检测技术平台对急性呼吸道感染疾病的病原微生物快速检测具有实用价值,尤其在解决流行病病原学分析方面具有重要意义。