Direct Fluorescent Antibody Method Research Articles

Comparative evaluation of some techniques used for the detection of rabies virus.

Neurotropic viruses in the family Rhabdoviridae, genus Lyssavirus, are what cause rabies, an acute, progressive, and highly lethal encephalomyelitis. Evaluation of the used diagnostic techniques to determine the most simple; rapid and accurate test for rabies virus (RABV) recognition in different specimens aiming to reach a rapid diagnosis as a step aid in the disease control and to prevent or even minimize the suspected hazard. The used techniques included an infection trial of Swiss mice with the mice-adapted challenge rabies virus followed by the detection of the virus in the infected mices' brains. Virus detection was carried out through the application BHK21 cell line infection; fluorescent antibody technique; latex agglutination test (LAT); direct enzyme-linked immunosorbent assay (ELISA); rabies antigen detection kit ELISA; conventional polymerase chain reaction (PCR). It was found that virus inoculation in mice and BHK21 cell lines needs 5-7 days with positivity of 90% and 100%, respectively. Rapid antigen kit was able to detect rabies antigen in mice brains suspension and BHK21 infected fluid within 3-5 minutes with percentages of 60% and 55.5%, respectively. In 1-1.5 hours, the direct fluorescent antibody method (DFAT) detected 90% and 100% of the rabies antigen in BHK21 cell line infection and brain impressions, respectively. Latex agglutination showed clear results with 88.8% with BHK21 infected fluid within 3-5 minutes while it did not carry out on brain emulsions to prevent falsely positive results brought on by the presence of tissue fragments. Conventional one-step PCR revealed 100% positivity with either brain or cell culture preparations within 2 days. Direct ELISA showed 88.8% positivity with BHK21 infected fluid with 1 day of work. Mice inoculation test, cell culture infection; DFAT and PCR are the most accurate techniques for the detection of RABV with a positivity of 90%-100% followed by LAT and ELISA with a positivity of 88.8%, and lastly, rabies antigen ELISA kit (RAK) with a positivity of 55.5%-60% taking in consideration the required time for each. In addition, the positivity % of the applied tests revealed their sensitivity and specificity.

Comparison among Modified Acid-Fast Stain and Some Immunological Methods in Diagnosis of Cryptosporidium parvum in Kut City

A total of 100 children who attending to Al-Karamah Teaching Hospital at Kut city were suffered from watery diarrhoea and abdominal pain were examined. The present study was conducted from October 2011 to January 2012. A thirty-four stool samples in which Cryptosporidium parvum oocysts had been seen by modified acid –fast stain examination were investigated, positivity detected with the ImmunoCard method, and Direct Fluorescent Antibody( DFA ) method was found to be (30%), (28%) and (34%) respectively.Children aged one month to 144 months presenting with acute or persistent diarrhoea were selected randomly. prevalence of Cryptosporidium parvum infection was significantly higher (59%) in children under one year old compared to children between (7-12) years old (4%). The infective rate of Cryptosporidium parvum in males was higher 17% than females 13% . Also, there was no significant difference among children in gender.

Serotyping of Legionella Bacteria Isolated from Various Water Systems in the Central Anatolia Region

Objective: Legionella bacteria are waterborne environmental pathogens that are considered a public health problem because they cause Legionnaires' disease, which is a nationally notifiable disease. Materials and Methods: Legionella analysis was performed in a total of 651 water samples collected during the years 2015 (450) – 2016 (201). Water samples were collected from hospitals (64.66%), hotels (15.05%), the automotive industry (14.43%) and from the buildings (5.83%) in the Central Anatolia Region. After the isolation of Legionella by the filtration and culturing method, serogroup and subtypes were determined via latex agglutination tests and the direct fluorescent antibody method. Results: In 2015, the Legionella positivity rate was 8.6%, where 28.2% of detections were from L. pneumophila serogroup-1. Six isolates were found to be Philadelphia, four were Olda, and one was Bellingham subgroup. Overall, 64.1% were L. pneumophila serogroup 2-14. Moreover, 14 isolates were SG-5, 10 were SG-6, and one was SG-10. 7.7% were unidentified Legionella species. The Legionella species identified were L. micdadei and L. longbeachae. In 2016, the Legionella positivity rate was 10.4%, with 28.6% of them being from L. pneumophila serogroup-1. Four were found to be Olda and two were Philadelphia subgroups. Overall, 66.6% of them were L. pneumophila serogroup 2-14. Moreover, six of them were SG-5, four were SG-6, and four were SG-2. 4.7% were unidentified Legionella species. There was only one species detected as L. micdadei. Conclusions: It has been observed that the distribution of Legionella has exhibited diversity in different water systems throughout the Central Anatolia Region.

723. Cryptosporidium Detection in Preserved Stool Specimens: A Comparison Study of EIA, DFA, and Direct Microscopic Method

Background Cryptosporidium is an intestinal parasite that may cause diarrhea. Laboratory diagnosis largely relies on microscopic or immunology-based antigen detection. Direct fluorescent antibody (DFA) is considered the gold standard. Enzyme immunoassay (EIA) is an attractive alternative, but direct comparison studies for the performance together with the impact from different specimen preservation media are limiting. MethodsWe compared these three methods for the detection of Cryptosporidium oocysts (direct microscopic) or antigen (DFA or EIA) from stool samples preserved in either 10% buffered formalin, Cary-Blair/C&S, or Total Fix (MCC, Torrance, CA). The DFA from Meridian Bioscience (Cincinnati, OH) and the EIA using CRYPTOSPORIDIUM II (TechLab®, Blacksburg, VA) were performed according to the manufacturer’s instructions. The direct microscopic method was performed according to laboratory protocols, including direct wet mount, modified acid-fast stain, or permanent trichrome stain. ResultsA total of 140 samples, including 116 clinical specimens, 20 validation panel samples and 4 proficiency survey specimens, were examined (Table 1). The DFA and EIA methods produced 100% concordant results using all three preservatives, while the microscopic method had decreased sensitivity. All microscopic positives remained positive for both the DFA and EIA. Cross-reactivity from other parasites, such as Giardia, of the two immunoassays was not observed. ConclusionWhile the two immunological methods both outperformed the microscopic method, the EIA has the advantages of being objective, simple to perform, has less hands-on time, and thus makes it an attractive option for high throughput Cryptosporidium detection. Disclosures Kileen L. Shier, PhD, D(ABMM), MLS(ASCP)CM, Quest Diagnostics (Employee)

Etiology of Severe Pneumonia in Children in Alveolar Lavage Fluid Using a High-Throughput Gene Targeted Amplicon Sequencing Assay.

Objective: To evaluate the diagnostic value of a high-throughput gene targeted amplicon sequencing (TAS) assay for detecting pathogenic microorganisms in alveolar lavage fluid (ALF) from children with severe community-acquired pneumonia (SCAP).Methods: A retrospective study was performed on 48 frozen ALF samples from 47 severe pneumonia cases admitted to Children's Hospital of Fudan University from January 1, 2019, to March 31, 2019. All samples were tested by a multiplex PCR (Multi-PCR) assay and a TAS assay. The results of the TAS panels were parallel compared with Multi-PCR and Conventional Tests (CT) including culture, direct fluorescent antibody method (DFA), and singleplex polymerase chain reaction (PCR).Results: The proportion of pathogens detection by CT was 81.2% (39/48). The 8 common respiratory viruses including respiratory syncytial virus (RSV), adenovirus (ADV), influenza A virus (FLUA), influenza B virus (FLUB), parainfluenza virus 1–3 (PIV1-3), and human Metapneumovirus (hMPV) were found in 31.2% (15/48) of the 48 samples by DFA. With the criteria of CT results used as “Golden Standard” for determing of TAS results, the proportion of pathogens detection by TAS was 70.8% (34/48). The difference of proportion of pathogens detection between TAS and CT was not statistically significant (p = 0.232). The sensitivity and specificity of TAS for pathogens detection based on CT were 87.1% (95% CI, 71.77–95.18%) and 100.0% (95% CI, 62.88–100%), the positive predictive value (PPV) and negative predictive value (NPV) were 100.0% (95% CI, 87.35–100%) and 64.2% (95% CI, 35.62–86.02%), respectively. While Multi-PCR results were used as “Golden Standard,” the total pathogens detection rate of TAS was 83.3% (40/48), which had a significant difference with that of Multi-PCR (p = 0.003). The sensitivity and PPV of TAS compared with Multi-PCR were 83.3% (95% CI, 69.23–92.03%) and 100.0% (95% CI, 89.08–100%), respectively. High rates of co-infection were proved by CT, Multi-PCR, and TAS. Mycoplasma pneumoniae (MP) and ADV were the two most frequently detected pathogens in all three assays.Conclusion: Compared with the CT and Multi-PCR methods, this TAS assay had a good performance in detecting bacteriological and viral pathogens from ALF. More research is needed to establish interpretation criteria based on TAS reads or analysis platforms.

Evaluation of patients with dry eye disease for conjunctival Chlamydia trachomatis and Ureaplasma urealyticum.

To determine the possibility of the development of dry eye disease (DED) as a result of persistent infection with Chlamydia trachomatis and Ureaplasma urealyticum in the conjunctiva of patients. This study was conducted on 58 patients of age range 20-50y, diagnosed with DED confirmed by Schirmer I test and tear breakup time. The non-dry eye control group included 27 subjects of the same age. Ocular specimens were collected as conjunctival scrapings and swabs divided into three groups: the first used for bacterial culture, the second and third taken to detect Chlamydia trachomatis and Ureaplasma urealyticum by direct fluorescent antibody (DFA) assay and polymerase chain reaction (PCR) method. Chlamydia trachomatis was detected in 65.5% and 76% of DED patients by DFA and PCR methods respectively. Ureaplasma urealyticum was found in 44.8% of DED infected patients using the PCR method. Both organisms were identified in only 37.9% of DED patients found to be infected. Control subjects had a 22% detection rate of Chlamydia trachomatis by DFA assay versus a 7% detection rate by PCR; while Ureaplasma urealyticum was detected in 3.7% of the controls by PCR method. The conjunctival culture revealed that gram positive microorganisms represented 75% of isolates with coagulase negative Staphylococci the most common (50%) followed by Staphylococcus aureus (20%), whereas gram negative microorganisms occurred in 25% of cases, isolating Moraxella spp. as the most frequent organism. Our results tend to point out that Chlamydia trachomatis and Ureaplasma urealyticum were detected in a moderate percentage of patients with DED, and could be a fair possibility for its development. PCR is more reliable in detecting Chlamydia trachomatis than DFA technique. The presence of isolated conjunctival bacterial microflora can be of some potential value.

Investigation of the presence of Blastocystis spp. in stool samples with microscopic, culture and molecular methods

Blastocystis species are enteric protozoa frequently detected in human and animals. Seventeen subtypes (STs) have now been identified, nine of them isolating from humans. The pleomorphic structure and genetic diversity of Blastocystis spp. and the absence of standardized diagnostic methods complicate the evaluation of current data. Microscopic methods such as native-lugol and trichrome staining are most frequently used methods in routine diagnosis, while culture and molecular methods are preferred for research purposes. The aims of this study were to investigate the presence of Blastocystis spp. in the stool samples of patients with gastrointestinal complaints by microscopic and culture methods, and to detect the subtypes of isolates by polymerase chain reaction (PCR). A total of 350 stool samples collected from patients with diarrhea (n= 157) and without diarrhea (n= 193) were included in the study. Presence of Blastocystis spp. in the samples were investigated by native-lugol examination, trichrome staining and direct fluorescent antibody (DFA) methods. Ringer's solution containing 10% horse serum and 0.05% asparagine was used for cultivation. The cultures were evaluated after 3-4 days of incubation at 37°C by microscopic examination. The subtypes of Blastocystis spp. strains isolated from the cultures have been identified by PCR using sequence-tagged site primers. A total of 66 (19%) stool samples, of them 26 (16.6%) were from diarrheal and 40 (21%) from non-diarrheal cases, yielded Blastocystis sp. growth in culture. Among the evaluated samples, 12% (42/350) were found positive with native-lugol examination, 17% (58/350) with trichrome staining, and 19% (66/350) with DFA method. The agreement of culture and native-lugol method was estimated as strong (κ= 0.752), while it was very strong between culture with trichrome staining and DFA methods (κ= 0.922 and κ= 1.00, respectively). When the culture was accepted as reference method, the sensitivity and specificity rates of native-lugol method were 65% and 100%, trichrome staining method were 88% and 100%, and DFA method were 100% and 100%, respectively. Forty-three (65%) of Blastocystis spp. positive samples were subtyped by PCR, while 23 isolates could not be subtyped. The most frequent detected subtype was ST3 (12/43; 28%), followed by ST1 (6/43; 13.9%), ST4 (5/43; 11.6%) and ST7 (5/43; 11.6%), ST2 (3/43; 7%) and ST6 (1/43; 2.3%). ST5 was not detected in this study and 11 (25.6%) samples have been identified to have mixed subtypes. The differences of Blastocystis spp. positivity rates and the distribution of the subtypes between the patients with or without diarrhea were not found statistically significant (p> 0.05). In our study, ST3 was the most frequently identified Blastocystis spp. subtype, similar to the previous national studies, however ST6 and ST7 have been identified for the first time. In conclusion, as the sensitivity of native-lugol examination is low, culture is time-consuming and laborious and PCR methods are costly and non-standardized, rapid, practical and high sensitive DFA is considered as the favourable method in the diagnosis of Blastocystis spp. in routine laboratories.

Role of Polyphosphate Kinase Gene (ppk) for Survival of Vibrio cholerae O1 in Surface Water of Bangladesh

Polyphosphate provides a substitute for ATP and energy source when phosphorus is a limiting resource in nature. The present study focuses on the role ofpolyphosphate for the survival of Vibrio cholerae in the aquatic habitats as an autochthonous bacterium. The survival advantages of polyphosphate of V. cholerae O1 having (wild type) and lacking (mutant) polyphosphate kinase (ppk) gene in surface water and with Anabaena variabilis were compared by cultural, Direct Fluorescent Antibody (DFA) and polymerase chain reaction methods in natural water microcosms. The microcosm's water was prepared by filtering and physicochemical parameters were also investigated by standard methods. The results revealed that both fresh and saline water, the wild type strain enhanced survival in cultural conditioned than ppk mutant strain. However, Fluorescent Antibody Direct Viable Counts (FADVC) and Polymerase Chain Reaction (PCR) results noted both strains have the equal survival strategy in viable but nonculturable state (VNC). In conclusion, it could be hypothesized that the polyphosphate inclusion body might keep cultivable and survivable at low phosphate natural environment of the aquatic bacterium.

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.

A simple biological marker to differentiate the types of Herpes Simplex Viruses in resource-limited settings

Herpes simplex viruses (HSV) multiply readily on the chorioallantoic membrane (CAM) of embryonated hen's egg and produce easily visible foci or pocks on this membrane. In the present study, pocks produced by the two antigenic types of HSV (1 & 2) were compared to evaluate the effectiveness of typing HSV isolates by pock size on CAMs. A total of 57 HSV isolates from both non-genital and genital samples were typed by the pock size produced on the CAMs of fertile hen's eggs. Twenty two HSV isolates yielded visible pocks on CAM, of which 9 (40.9%) produced small pocks, while 13 (59.1%) produced large pocks. All pocks produced on CAM were confirmed by antigenic typing by the Direct Fluorescent Antibody (DFA) method. HSV isolates which produced small pocks were in complete (100%) concordance with HSV type-1, while those producing larger pocks were in full (100%) concordance with HSV type-2. Thus, the pock size on CAM of embryonated fertile hen's egg may be used as a simple and relatively inexpensive biological marker for the differentiation of HSV types 1 & 2.

A survey of <i>Vibrio cholerae</i> O1 and O139 in estuarine waters and sediments of Beira, Mozambique

This study determined whether the estuarine and freshwater environment in Beira, Mozambique, serves as a reservoir of Vibrio cholerae O1 and O139. Ninety-nine estuarine water samples were collected at 6 sites in Beira. An additional 54 samples were collected from rural areas around Beira which included 3 freshwater lake samples, 15 river, 5 pond, and 4 estuarine water samples, and an equivalent number of sediment samples, collected from the same sites as the water samples. In addition, fish scales from 5 ocean fish and 1 deep sea water sample were also collected. The samples were analysed for the presence of V. cholerae O1 and O139 using culture methods, the direct fluorescent antibody (DFA) method and polymerase chain reaction (PCR) using a single-primer pair for the ompW gene and a semi-nested PCR selecting for the ctxAgene, encoding subunit A of cholera toxin. DFA results showed 37 V. cholerae O1- and 6 O139-positive samples. Vibrio cholerae O1 and O139 were observed on the scales of 4 of the 5 fish. The findings of the study provided in situ evidence for V. cholerae O1 and O139, predominantly as viable but non-culturable cells in the aquatic environment of Beira. This is the first record of the presence of V. cholerae O139 in the estuarine environment on the coast of Africa.Keywords: Vibrio cholerae O1, Vibrio choleare O139, water quality, estuary, PCR, DFA

Limited Applicability of Direct Fluorescent-Antibody Testing for Bordetella sp. and Legionella sp. Specimens for the Clinical Microbiology Laboratory

The rapid diagnosis of infections with Bordetella and Legionella species is important for patient management. With observed increases in direct fluorescent-antibody (DFA) testing volumes, we retrospectively compared the performance characteristics of DFA testing to those of culture and PCR. For Bordetella sp., samples were classified as positive by DFA testing (184 [3%] of 6,195 samples) and culture (150 [2%] of 6,251 samples) significantly less often than by PCR (2,557 [10%] of 26,929 samples). Of 360 samples tested by both DFA and PCR methods, 81 (16 by DFA testing and 79 by PCR) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 18% and 99%, respectively. Of 1,426 samples tested by both DFA and culture methods, 48 (44 by DFA testing and 15 by culture) were determined to be positive for Bordetella, with a sensitivity and specificity of DFA testing of 73% and 98%, respectively. For Legionella sp., samples were identified as positive by DFA testing (31 [0.25%] of 12,597 samples) and culture (85 [0.6%] of 13,572 samples) significantly less often than by PCR (27 [4%] of 716 samples). Of 62 samples tested by both DFA and PCR methods, none were positive for Legionella sp. by DFA testing and 3 were positive by PCR. Of 3,923 samples tested by both DFA and culture methods, 22 (3 by DFA testing and 21 by culture) were positive for Legionella sp., with a sensitivity and specificity of DFA testing of 9.5% and 100%. Overall, DFA testing for Bordetella sp. and Legionella sp. is an insensitive method, and despite its continued popularity, clinical microbiology laboratories should not offer it when more sensitive tests like PCR are available.

Seasonal cholera caused by Vibrio cholerae serogroups O1 and O139 in the coastal aquatic environment of Bangladesh.

Since Vibrio cholerae O139 first appeared in 1992, both O1 El Tor and O139 have been recognized as the epidemic serogroups, although their geographic distribution, endemicity, and reservoir are not fully understood. To address this lack of information, a study of the epidemiology and ecology of V. cholerae O1 and O139 was carried out in two coastal areas, Bakerganj and Mathbaria, Bangladesh, where cholera occurs seasonally. The results of a biweekly clinical study (January 2004 to May 2005), employing culture methods, and of an ecological study (monthly in Bakerganj and biweekly in Mathbaria from March 2004 to May 2005), employing direct and enrichment culture, colony blot hybridization, and direct fluorescent-antibody methods, showed that cholera is endemic in both Bakerganj and Mathbaria and that V. cholerae O1, O139, and non-O1/non-O139 are autochthonous to the aquatic environment. Although V. cholerae O1 and O139 were isolated from both areas, most noteworthy was the isolation of V. cholerae O139 in March, July, and September 2004 in Mathbaria, where seasonal cholera was clinically linked only to V. cholerae O1. In Mathbaria, V. cholerae O139 emerged as the sole cause of a significant outbreak of cholera in March 2005. V. cholerae O1 reemerged clinically in April 2005 and established dominance over V. cholerae O139, continuing to cause cholera in Mathbaria. In conclusion, the epidemic potential and coastal aquatic reservoir for V. cholerae O139 have been demonstrated. Based on the results of this study, the coastal ecosystem of the Bay of Bengal is concluded to be a significant reservoir for the epidemic serogroups of V. cholerae.

Evaluation of seven commercial antigen detection tests for Giardia and Cryptosporidium in stool samples

Stool samples from patients with abdominal symptoms were used to evaluate different copro-diagnostic assays for the detection of Giardia and Cryptosporidium. Results from microscopical examination following conventional stool concentration and direct fluorescent-antibody methods were compared with various commercially available immunochromatographic and enzyme immunoassays. Of 220 samples, 45 were positive for Giardia and 17 for Cryptosporidium. For Giardia, the sensitivities obtained by Ridascreen Giardia, Rida Quick Giardia, Rida Quick Combi and Giardia-Strip were 82%, 80%, 80% and 44%, respectively. For Cryptosporidium, the sensitivities obtained by Rida Quick Cryptosporidium, Ridascreen Cryptosporidium, Rida Quick Combi and Cryptosporidium-Strip were 88%, 82%, 82% and 75%, respectively. The specificity of all tests was > or = 98%. Other intestinal parasites were present in 68 samples, but cross-reactions with other protozoan or helminthic parasites were not observed. Overall, the copro-antigen assays were less time-consuming and easier to perform, but were less sensitive than conventional microscopical methods. Thus, these tests might be a useful addition to, but not a substitute for microscopical methods in the diagnosis of travel-associated giardiasis and cryptosporidiosis.

A Comparison of Induced and Expectorated Sputum for the Diagnosis of Pneumocystis carinii Pneumonia

To compare the sensitivities of expectorated and induced sputum for the diagnosis of Pneumocystis carinii pneumonia (PCP). Retrospective review. Academic medical center. Forty-five patients diagnosed as having PCP who had direct fluorescent antibody testing for P carinii on either expectorated or induced sputum. Patients were stratified according to the method of sputum production (induced vs expectorated). The two groups were similar with respect to demographic characteristics, use of prophylaxis with aerosolized pentamidine, serum lactate dehydrogenase level, and arterial oxygen level. When only the initial sputum for each patient was analyzed, there was a similar sensitivity of induced sputum, positive in 10 of 18 samples (56%), and expectorated sputum, positive in 14 of 27 samples (52%) (p>0.05). There was no significant difference in the sensitivity of induced and expectorated sputum for the diagnosis of PCP when the direct fluorescent antibody method of staining was used.

Genital Chlamydia trachomatis (serotypes D-K) infection in Jamaican commercial street sex workers.

OBJECTIVES: To determine the prevalence of genital Chlamydia trachomatis infections in commercial street sex workers (CSSW) in Jamaica. METHODS: The prevalence of C trachomatis infection was determined in 129 Jamaican...

Diagnosis of Legionella pneumophila Infection by Polymerase Chain Reaction

We examined the application of the polymerase chain reaction (PCR) for the diagnosis of legionellosis. Eight intratracheal aspirates were collected from a patient with pneumonia caused by Legionella pneumophila serogroup 2, and serial 10-fold dilutions of the samples were obtained. Two samples were positive for L. pneumophila by the direct fluorescent antibody (DFA) method down to a 10(-2) concentration, and one was positive down to a 10(-4) concentration. PCR was positive for all eight samples, and the sensitivity was greater than that of the DFA method. Only one of the eight samples yielded organisms in culture: the L. pneumophila serogroup 2 strain was isolated on buffered charcoal yeast extract alpha agar as an atypical white, papillate colony.

Detection of Legionella species in reclaimed water and air with the EnviroAmp Legionella PCR kit and direct fluorescent antibody staining

Reclaimed water is an important resource for areas with inadequate water supplies. However, there have been few studies on the variety of microorganisms found in this type of water, since typically reclaimed water is examined only for the presence of coliform bacteria. Many microorganisms, including the legionellae, are known to be more resistant to chlorine than are coliform bacteria. Previously, we detected > 10(3) Legionella cells per ml in primary and secondary sewage effluents and observed no significant reduction in population numbers throughout the treatment process. In this study, we detected Legionella spp. in chlorinated effluent by using an EnviroAmp Legionella PCR kit and direct fluorescent antibody (DFA) staining. However, we were not able to isolate Legionella spp. from either natural or seeded reclaimed water samples. This suggests that the Legionella spp. detected by the PCR and DFA methods may be injured or viable but nonculturable after exposure to the high residual chlorine levels typically found in this type of water source. The numbers of coliform bacteria were low (< 2 cells per 100 ml) in most reclaimed water samples and were not correlated with the presence or absence of Legionella spp. We also collected air samples from above a secondary aeration basin and analyzed them by using the PCR, DFA, and plate culture methods. Legionella spp. were detected in the air obtained from above the secondary basin with all three methods. We concluded that the PCR was superior to the culture and DFA methods for detecting Legionella spp. in environmental water samples.

Disparity of Borrelia burgdorferi infection rates of adult Ixodes dammini on deer and vegetation.

Rates of infection with Borrelia burgdorferi were compared in adult Ixodes dammini ticks collected from deer at one coastal and two island sites with those collected from vegetation at the same sites. Ticks were examined using polyclonal direct fluorescent antibody. Spirochetes were observed in 47% of 288 ticks from vegetation as opposed to 13% of 276 ticks from deer (chi 2, P < .001). This disparity was increased when only male ticks were compared. Among female ticks from deer, the infection rate was higher in flat than in engorged ticks. These findings may reflect spirochete loss from ingestion of borreliacidal antibodies in deer blood or may result from factors related to the sensitivity of direct fluorescent antibody methods. They indicate that erroneously low estimates of regional risk for Lyme disease may be obtained if ticks removed from deer are included in the determination of tick infection rates.

Colonization of broiler chickens by waterborne Campylobacter jejuni.

Chickens on a broiler farm in southern England were found to be colonized with Campylobacter jejuni of a single serotype, Lior 1 Penner 4. The farm was the sole supplier of a local slaughterhouse associated with a campylobacter outbreak in 1984 caused by this serotype. The serotype persisted on the farm for at least 18 months after the outbreak; its prevalence in the human population served by the farm remained high until it disappeared from the farm in 1986. The possible sources and routes of transmission of C. jejuni to the broilers on the farm were investigated. The results showed that vertical transmission, feed, litter, small mammals, and environmental or airborne cross-contamination between sheds or successive crops could be excluded as persistent sources of C. jejuni. The predominant source of C. jejuni on the farm was shown to be the water supply. Direct microscopy and fluorescent antibody methods revealed presumptive campylobacters throughout the farm's water system. Campylobacter-free chickens raised in an animal house and given water from the farm supply became colonized with the serotype of C. jejuni endemic on the farm (Lior 1 Penner 4). An intervention program based on water chlorination, shed drinking system cleaning and disinfection, and withdrawal of furazolidone from feed reduced the proportion of birds colonized with campylobacter from 81 to 7% and was associated with a 1,000- to 10,000-fold reduction in campylobacters recoverable from the carcasses. Two months after the end of the intervention program colonization of the birds returned to high levels (84%), indicating that there was a temporal association between intervention and reduced colonization with C. jejuni. Investigations continue to establish the general applicability of these findings.