用聚合酶链反应（PCR）检测支气管肺泡灌洗（BAL）液中的嗜肺军团杆菌

本文研究了由嗜肺军团杆菌的巨噬细胞感染性增效子（ｍｉｐ）基因设计的一对引物,用ＰＣＲ扩增嗜肺军团杆菌３、５、７、８血清型的４个标准菌株的特异ＤＮＡ序列,研究了用该引物扩增ＢＡＬ液中嗜肺军团菌特异ＤＮＡ序列的方法、灵敏度及特异性。结果表明：用上述引物扩增嗜肺军团菌４个标准菌株的ＤＮＡ,均可得到２０７ｂｐ的特异扩增产物,ＢＡＬ液中的军团菌经离心及裂解液裂解后,可直接进行ＤＮＡ扩增,当ＢＡＬ与液中军团菌量为２×１０３ＣＦＵ／ｍｌ时,即可检测出特异扩增带（电泳法）,除军团菌外,其它受试细菌均无此特异性扩增,用本法对４２例临床非典型肺炎患者的ＢＡＬ液进行嗜肺军团菌的检测,在４２份嗜肺军团菌培养均为阴性的ＢＡＬ液中,其中一例ＰＣＲ检测军团菌为阳性。本研究提示：用ＰＣＲ检测ＢＡＬ液中的军团菌是可行的,并有快速、灵敏、特异之忧点。     

Detection of cytomegalovirus in infant cerebrospinal fluid by conventional PCR, nested PCR and PCR-Digene

The possibility of amplification of human cytomegalovirus (HCMV) DNA in cerebrospinal fluid (CSF) for the diagnosis of HCMV central nervous system (CNS) infection in infants was studied. Single-step PCR, nested PCR and PCR-Digene were used to assay CSF specimens from 37 patients. Criteria for patient inclusion in the study were: 1. clinical manifestations suggesting CMV neuroinfection such as seizures, hypertonia, hypotonia, intracranial calcification, microcephaly, chorioretinitis; 2. any of the following symptoms: anaemia, hepetomegaly, prolonged cholestatic jaundice, or hepatitis, splenomegaly, thrombocytopenia, intrauterine hypotrophy; 3. serologic presentation, and/or positive results for CMV infection obtained by single-step PCR and PCR-Digene in urine and/or blood. PCR-Digene results were positive in 6 CSF samples. Four CSF samples were positive by nested PCR and 1 CSF sample by single step PCR. We found that the double PCR was about ten or more times more sensitive than single PCR and the PCR-Digene was only three times more sensitive than nested-PCR. The results were correlated with serology. Thirty-three out of 37 examined patients were seropositive (ELISA IgG); ELISA IgM gave positive results in 9 patients. In control studies, cells infected with other members of the herpes virus family were negative with these methods, which suggest that amplification combined with primers from the IE and the L-region of CMV is specific. In conclusion, nested-PCR seems to be the best method for early diagnosis of CMV infection in CSF due to an absence of false positive results and its high specificity and sensitivity.     

中药大蒜素体外抗弓形虫效应及其机制的研究

目的 观察大蒜提取物体外抗弓形虫的作用效果及其机制.方法 将弓形虫RH株速殖子与兔肾细胞共同培养,加入不同浓度的大蒜素(实验组)和磺胺嘧啶(阳性对照组),培养不同时间后取出细胞,固定染色后观察细胞感染率及每个纳虫泡中弓形虫速殖子的数量;采用MTT比色法观察大蒜素对弓形虫速殖子侵袭细胞及其正常细胞增殖的影响;采用台盼兰着色法观察大蒜素对弓形虫速殖子活性的影响;采用TUNEL末端标记法检测弓形虫速殖子凋亡率,对药物的效应和机制进行探讨.结果 (1)10～80 μg/ml的大蒜素能抗弓形虫的感染,呈现剂量依赖性,与时间无明显的相关性.(2)40 μg/ml、80 μg/ml的大蒜素不能抑制细胞的增殖,对细胞无明显的毒副作用;160 μg/ml的大蒜素对细胞有明显的毒副作用.(3)大蒜素80 μg/ml时,台盼兰着色率最高,弓形虫的活力最低.(4)大蒜素80 μg/ml的浓度时,弓形虫速殖子凋亡率最高.结论 大蒜素可以抑制弓形虫速殖子的活力、对宿主细胞的感染力、在细胞内的增殖,无明显的毒副作用,最适宜浓度为80 μg/ml.大蒜素是一种良好的抗弓形虫药物,诱导弓形虫速殖子凋亡是其抗弓形虫机制之一.     

DNA persistence after treatment of Lyme borreliosis

One hundred twenty-four patients—53 with neuroborreliosis, 48 with erythema migrans, and 23 with Lyme arthritis—were tested in a prospective study for the presence of the DNA of Borrelia burgdorferi sensu lato in plasma, cerebrospinal fluid (CSF), urine, and synovial fluid by nested polymerase chain reaction (PCR). Specific DNA was detected using five amplification systems simultaneously: three targeted chromosomal genes encoding 16S rDNA, flagellin, and p66; and two plasmid sequences of OspA and OspC. Patients were examined clinically and by PCR before and after treatment and again after 3 and 6 months. Before treatment, the specific DNA was detected in 78 patients (62.9 %). Forty-one neuroborreliosis patients were DNA-positive (77.4 %), with CSF positivity in 26 patients, urine in 25, and plasma in 16. Twenty-six erythema migrans patients were DNA-positive (54.2 %), with plasma positivity in 18 cases and urine in 14. Eleven Lyme arthritis cases (47.8 %) were DNA positive (six in urine, five in plasma, and four in synovial fluid). The frequency of PCR positives was comparable in CSF and urine, and it was lower by approximately 50 % in plasma. Specific DNA was also found in a significant number of patients in later testing periods: 48 patients after treatment, 29 patients after 3 months, and 6 patients after 6 months. The prolonged PCR positivity was not explainable by persistent infection according to the clinical manifestations of the disease. Possible explanations of the problem are discussed.     

弓形虫（Toxoplasma gondii）的PCR检测方法在笼养猕猴群中的应用

目的建立快速、灵敏、特异及检测结果易判断的PCR方法,并应用于大规模猕猴种群的弓形虫常规检测中。同时比较巢式PCR和单一PCR的一致性。方法根据弓形虫保守基因p30（SAG1）设计了内、外两对进行巢式PCR扩增以及B1基因设计一对引物进行单一PCR扩增,将DNA样本进行10倍倍比稀释,以检测巢式PCR反应的灵敏度;并对医学生物学研究所自繁猕猴共150只进行了弓形虫检测。结果巢式PCR检测法检测限度可达10^-3ng/uL,而且方法特异。两种PCR法检测结果基本一致,其中巢式PCR检测阳性率（10％）稍高于单一PCR检测阳性率（8．67％）。结论巢式PCR和一次PCR方法都可应用于猕猴弓形虫的常规检测中,并提示巢式PCR比单一PCR更敏感、检出率更高。     

Detection of Candida species by polymerase chain reaction (PCR) in blood samples of experimentally infected mice and patients with suspected candidemia

In this study, we have established and evaluated a genus-specific polymerase chain reaction (PCR) and species-specific nested PCRs for the detection of Candida species in blood samples of neutropenic mice and patients suspected of candidemia. DNA segments of the gene encoding cytochrome P450 L1A1 were targeted for amplification by using genus and species-specific primers. As compared to the genus-specific PCR, the species-specific nested PCRs improved the sensitivity by 10 times with the detection limit < 10 yeast cells. Of the 18 blood samples tested daily over a period of 8 days following Candida albicans infection in neutropenic mice, four samples were positive by genus-specific PCR and 11 were positive by species-specific nested PCR. The PCR results were correlated with culture findings obtained on blood samples. Two of the three blood culture-positive samples were positive by genus-specific PCR and all the three with species-specific nested PCR. Among 15 mice, which were negative by blood culture but had C. albicans isolated from visceral organs, 2 and 8 mice yielded positive results by genus-specific PCR and species-specific nested PCR, respectively. Consistent with the results of the animal study, species-specific nested PCR yielded much higher positivity as compared to culture (52.2% versus 21.2%) in patients suspected for candidemia. Moreover, 8 specimens which were negative for Candida by genus-specific PCR became positive by species-specific nested PCR. No correlation was apparent between PCR positivity and Candida antigen titers. The results suggest that nested PCR is a sensitive technique for the detection of Candida species from blood samples, and thus it may have application in the diagnosis of suspected cases of candidemia and candidiasis.     

Physical and genetic mapping of cloned ribosomal DNA from Toxoplasma gondii: primary and secondary structure of the 5S gene.

The ribosomal DNA (rDNA encoding rRNA) of the obligately intracellular protozoan parasite, Toxoplasma gondii, was identified, cloned, physically mapped, its copy number determined, and the 5S gene sequenced. Using total RNA as a probe, a collection of recombinant lambda phages containing copies of rDNA were isolated from a lambda 2001 tachyzoite genomic library. Northern gel hybridization confirmed specific homology of the 7.5-kb rDNA unit, subcloned into pTZ18R, to T. gondii rRNA. The mapped rDNA found in pTOX1 contained small ribosomal subunit (SS; 18S)- and large ribosomal subunit (LS; 26S)-encoding genes localized using intragenic heterologous probes from the conserved sequences of the SS (18S) and LS (28S) Xenopus laevis genes. the physical mapping data, together with partial digestion experiments and Southern gel hybridization, confirmed a 7.5-kb rDNA unit arranged in a simple head-to-tail fashion that is tandemly repeated. We estimated the rDNA repeat copy number in T. gondii to be 110 copies per haploid tachyzoite genome. Parts of the SS gene and the complete 5S gene were sequenced. The 5S gene was found to be within the rDNA locus, a rare occurrence found only in some fungi and protozoa. Secondary-structure analysis revealed an organization remarkably similar to the 5S RNA of eukaryotes.     

Adapting a conventional PCR assay for Toxoplasma gondii detection to real-time quantitative PCR including a competitive internal control

We have developed a quantitative PCR assay (LightCycler* using the pair of primers JW58 and JW59 for the detection of the 35-fold repeated B gene of oxoplasma gondii. This real-time PCR, using fluorescence resonance energy transfert (FRET) hybridization probes, allows the quantification of . gondii with several technical requirements not previously described: i) an internal amplification control (co-amplified in a single tube with the same primers), ii) Uracil-N-Glycosylase and iii) a standard curve corresponding to a serial dilution from a calibrated suspension of T. gondii ranging from 40 to 4.106( )parasites in one ml of amniotic fluid (1 to 105( ) . gondii/PCR). In artificial samples, one parasite could be detected if at least three reactions were performed.     

AM真菌DNA的提取与PCR-DGGE分析

分别从丛枝菌根(AM)真菌的单孢、宿主植物根系及土壤样品中提取DNA,对AM真菌的18SrDNA中NS31/Glol区进行Nested PCR特异性扩增,表明Nested PCR能很好地以微量DNA为模板扩增出目标产物;对扩增产物进行DGGE电泳,3种样品表现出不同的DGGE指纹图谱特征。本文认为,将Nested PCR与DGGE技术相结合,可以成为AM真菌分子生态学研究的有效途径。     

Detection of human T-cell leukemia virus type I (HTLV-I) provirus in an infected cell line and in peripheral mononuclear cells of blood donors by the nested double polymerase chain reaction method: comparison with HTLV-I antibody tests.

Human T-cell leukemia virus type I (HTLV-I) provirus DNA from the cultured cell line HUT 102 and from peripheral mononuclear cells (PBMC) of anti-HTLV-I antibody-positive Japanese blood donors was detected by the nested double polymerase chain reaction (PCR) method. This procedure consists of a first amplification and a second amplification with the products of the first amplification and primers interior to the first primers. Using this method, we demonstrated that it is possible to detect single-template DNA. Polyacrylamide gel electrophoresis of the nested double PCR products, with our primers, revealed three bands with excess amounts of template DNA, two bands with moderate amounts, and a single band with limited amounts. The amount of provirus in PBMC was roughly estimated from the results of the nested double PCR. Particle agglutination (PA) assays and indirect immunofluorescence testing (IF) with mixed MT-2 cells and Molt-4 cells as targets to detect anti-HTLV-I antibody were performed, and the results were compared with those of the nested double PCR of the pX region. None of the 101 PA-negative samples were positive in either the IF or PCR test. Of the 155 samples that were antibody positive by the PA assay, 57 were positive by both PCR and IF. Furthermore, the results of the IF and PCR tests coincided completely. It was therefore concluded that the IF method is most appropriate for confirmation of the PA assay currently used in most diagnostic laboratories and blood centers.     

Horizontal transmission of Toxoplasma gondii in squirrel monkeys (Saimiri sciureus).

The possibility of horizontal transmission of T. gondii was examined in squirrel monkeys. After three monkeys were inoculated perorally with 1.1-2.1 x 10(3) cysts of the T. gondii ME49, the animals were divided into two cages and maintained with one normal monkey for each cage as a cagemate. Two out of the three T. gondii-inoculated monkeys died, and the remaining one monkey was sacrificed in a moribund state one week after infection because of acute toxoplasmosis. Many T. gondii tachyzoites were recovered from broncho-alveolar lavages and were also found histopathologically in the lung, liver, spleen, kidney and lymph nodes and impression smears of tissues from the three T. gondii-inoculated monkeys by Giemsa staining. Anti-T. gondii antibody was examined by immunoblot assay in these animals, and the antibody to T. gondii major surface membrane protein (p30) could be detected after the start of experiment. Furthermore, a specific band of T. gondii NTPase gene was observed by PCR in the liver and lung of infected and cagemate monkeys, and the sequence of the second PCR products obtained from the cagemates, which were clinically normal but gave a positive result in immunoblotting assay, was exactly the same as the sequence of the NTPase gene of T. gondii ME49. These findings suggested that transmission of T. gondii from the infected monkeys to cagemates occurred easily, and since many T. gondii tachyzoites were recovered from the bronchoalveolar lavages of the three T. gondii-inoculated monkeys, we suggest that aerosol infection plays an important role for the enzootic toxoplasmosis in colonies of squirrel monkeys.     

K24 T. gondii isolate is a hybrid and has the virulence of lineage I isolates

A permanently high virulence was found in tachyzoites of T. gondii K24 after serial passage in mice (90 passages during 324 days). Virulence tests revealed that a single tachyzoite of the 50th passage represented LD100 for mice. Analysis of genotype of K24 isolate was done by PCR/RFLP with ROP1/Ddel, SAG1/Ddel, 850/Rsal and IGS/Rsal and by RFLP/DNA with TGR1E sequence and Pstl enzyme. K24 isolate had an atypical genotype, with an association of type II (for ROP1, SAG1 genes and TGR1E sequence) and type I (for 850 gene) alleles, and a new pattern observed for IGS. All tested PCR/RFLP did not change through 2, 10, 20, 28, 40, 50, 60, 70, 81 and 90 tested passages. In RFLP/DNA with Pstl enzyme and TGR1E probe, K24 isolate produced a pattern with seven fragments of the size ranging from one to 23 kb and did not change through 7, 56, 70 and 83 tested passages. K24 T. gondii isolate is a hybrid and has the virulence of lineage I isolates.     

Loop-mediated isothermal amplification targeting the apxIVA gene for detection of Actinobacillus pleuropneumoniae

Loop-mediated isothermal amplification (LAMP) is a novel nucleic acid amplification method performed under isothermal conditions with high specificity and efficiency. We developed a diagnostic method based on LAMP for detection of Actinobacillus pleuropneumoniae . Using six specific primers targeting the apxIVA gene, the LAMP assay rapidly amplified the target gene within 30 min, requiring only a laboratory water bath for the reaction to occur. The resulting amplificon was visualized by adding SYBR Green I to the mixture. The results obtained from testing 15 A. pleuropneumoniae reference strains and other seven bacterial species strains showed that the LAMP was as specific as and 10 times more sensitive than nested PCR. Sixty-five tonsil samples were collected from 65 healthy pigs. All the samples were negative for A. pleuropneumoniae by immunomagnetic separation-based (IMS) bacterial isolation, nested PCR and LAMP, respectively. Meanwhile, 115 tonsil samples were also collected from 115 pigs with apparent respiratory problems. Twenty-two were positive by IMS bacterial isolation. All the samples that were positive by IMS bacterial isolation were also positive by nested PCR and LAMP. The LAMP assay demonstrated exceptionally higher sensitivity than nested PCR by picking up 14 additional positive cases (χ² test, P <0.0001); we concluded that LAMP was a highly sensitive and reliable method for detection of A. pleuropneumoniae infection.     

Invasive meningococcal disease and latex agglutination test — Is it still beneficial for diagnosis?

We showed current clinical usefulness of the latex agglutination (LA) test for confirmation of meningococcal etiology on 32 cerebrospinal fluid, 77 serum and 93 urine samples collected during the first week of hospitalization from 19 patients with laboratory confirmed invasive meningococcal disease. The positivity of the LA test in cerebrospinal fluid was 47%, in serum 42% and in urine 24%, while the PCR of cerebrospinal fluid and serum was positive in 95 and 47% cases, respectively. The latest positivity of the LA test was detected on day 2 in cerebrospinal fluid, on day 3 in serum and on day 4 in urine. In the group of patients who had received antibiotic therapy we found nonsignificant reduction of LA positivity and also statistically significant reduction of culture positivity in CSF (p = 0.04); the PCR positivity changed minimally. In blood samples, nonsignificant reduction of culture positivity and no difference in LA and PCR positivity was found. We did not find any statistically significant relationship between test results and clinical forms. The LA test can be therefore considered to be an auxiliary diagnostic method, rapid and easily practicable but less sensitive than PCR. It can be recommended especially for local laboratories where PCR is not available and the patient already received antibiotics before admission to the hospital.     

Molecular detection of Gluconacetobacter sacchari associated with the pink sugarcane mealybug Saccharicoccus sacchari (Cockerell) and the sugarcane leaf sheath microenvironment by FISH and PCR

Molecular tools for the detection of the newly described acetic acid bacterium Gluconacetobacter sacchari from the pink sugarcane mealybug, Saccharicoccus sacchari Cockerell (Homiptera: Pseudococcidae), and in the sugarcane leaf sheath microenvironment were developed. G. sacchari specific 16S rRNA-targeted oligonucleotide primers were designed and used in PCR amplification of G. sacchari DNA directly from mealybugs, and in a nested PCR to detect low numbers of the bacteria from sugarcane leaf sheath fluid and cane internode scrapings. A sensitivity level of detection of 40-400 cells/reaction was obtained using PCR from exponentially grown bacterial cultures and of 1-10 cells in cane internode scrapings and leaf sheath fluid samples using nested PCR. The specificity of the primer set was demonstrated by the lack of amplification product formation in PCR by closely related acetic acid bacteria, including Gluconacetobacter liquefaciens, and Gluconacetobacter diazotrophicus. A Cy3 labeled probe for G. sacchari was designed and shown to be specific for the species. Investigation of the mealybug microenvironment by whole cell fluorescent in situ hybridization revealed that G. sacchari appears to represent only a minor proportion of the population of the microbiota in the mealybugs tested. This study has shown the usefulness of 16S rRNA-based molecular tools in the identification and detection of G. sacchari from environmental samples and will allow these tools to be used in further ecological research.     

Detection of porcine circovirus type 1 in commercial porcine vaccines by loop-mediated isothermal amplification

A loop-mediated isothermal amplification (LAMP) method with a real-time monitoring system was developed for the detection of porcine circovirus type 1 (PCV1) in commercial swine vaccines. This method was highly specific for PCV1. No cross-reaction to porcine circovirus type 2, porcine parvovirus, pseudorabies virus, classical swine fever virus, and porcine reproductive and respiratory syndrome virus was observed. The analytical sensitivity of the LAMP for PCV1 DNA was 10 copies/μl in the case of positive recombinant plasmid comparable to that obtained from the nested polymerase chain reaction (nested PCR). Furthermore, 25 commercial swine vaccines were tested by both the LAMP and the nested PCR, and three of them were tested positive for PCV1 DNA. These results indicate that PCV1 DNA can be real-time detected by the LAMP; the method was highly specific, sensitive, and rapid for the detection of PCV1 DNA, particularly in commercial swine vaccines.     

Multiple displacement amplification of DNA from human colon and rectum biopsies: bacterial profiling and identification of Helicobacter pylori-DNA by means of 16S rDNA-based TTGE and pyrosequencing analysis

Amplifying bacterial DNA by PCR from human biopsy specimens has sometimes proved to be difficult, mainly due to the low amount of bacterial DNA present. Therefore, nested or semi-nested 16S rDNA PCR amplification has been the method of choice. In this study, we evaluate the potential use of whole genome amplification of total DNA isolated from human colon and rectum biopsy specimens, followed by 16S rDNA PCR amplification of multiple displacement amplified (MDA)-DNA. Subsequently, a H. pylori-specific 16S rDNA variable V3 region PCR assay was applied directly on MDA-DNA and, combined with pyrosequencing analysis; the presence of H. pylori in some biopsies from colon in patients with microscopic colitis was confirmed. Furthermore, temporal temperature gradient gel electrophoresis (TTGE) of 16S rDNA amplicons using primers flanking variable regions V3, V4, and V9, was used to establish bacterial profiles from individual biopsies. A variation of the bacterial profiles in the colonic mucosa in microscopic colitis and in normal rectal mucosa was observed. In conclusion we find the MDA technique to be a useful method to overcome the problem of insufficient bacterial DNA in human biopsy specimens.     

Efficacy of ponazuril in vitro and in preventing and treating Toxoplasma gondii infections in mice

Toxoplasma gondii is an important apicomplexan parasite of humans and other warm-blooded animals. Ponazuril is a triazine anticoccidial recently approved for use in horses in the United States. We determined that ponazuril significantly inhibited T. gondii tachyzoite production (P < 0.05) at 5.0, 1.0, or 0.1 microg/ml in African green monkey kidney cells. We used outbred female CD-1 mice to determine the efficacy of ponazuril in preventing and treating acute toxoplasmosis. Each mouse was subcutaneously infected with 1,000 tachyzoites of the RH strain of T. gondii. Mice were weighed daily, and ponazuril was administered orally in a suspension. Mice given 10 or 20 mg/kg body weight ponazuril 1 day before infection and then daily for 10 days were completely protected against acute toxoplasmosis. Relapse did not occur after prophylactic treatments were stopped. Toxoplasma gondii DNA could not be detected in the brains of these mice using polymerase chain reaction (PCR). One hundred percent of mice treated with 10 or 20 mg/kg ponazuril at 3 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Sixty percent of mice treated with 10 mg/kg ponazuril at 6 days after infection and 100% of mice treated with 20 mg/kg or 50 mg ponazuril 6 days after infection and then daily for 10 days were protected from fatal toxoplasmosis. Relapse did not occur after treatments were stopped. Toxoplasma gondii DNA was detected in the brains of some, but not all, of these mice using PCR. The results demonstrate that ponazuril is effective in preventing and treating toxoplasmosis in mice. It should be further investigated as a safe and effective treatment for this disease in animals.     

Development and amplification of multiple co-dominant genetic markers from single spores of arbuscular mycorrhizal fungi by nested multiplex PCR

Multiple co-dominant genetic markers from single spores of the arbuscular mycorrhizal (AM) fungi Glomus mosseae, Glomus caledonium, and Glomus geosporum were amplified by nested multiplex PCR using a combination of primers for simultaneous amplification of five loci in one PCR. Subsequently, each marker was amplified separately in nested PCR using specific primers. Polymorphic loci within the three putative single copy genes GmFOX2, GmTOR2, and GmGIN1 were characterized by sequencing and single strand conformation polymorphisms (SSCP). Primers specific for the LSU rDNA D2 region were included in the multiplex PCR to ensure correct identification of the Glomus spp. spores. Single AM fungal spores were characterized as multilocus genotypes by combining alleles of each amplified locus. Only one copy of each putative single copy gene could be amplified from each spore, indicating that spores are homokaryotic. All isolates of G. mosseae had unique genotypes. The amplification of multiple co-dominant genetic markers from single spores by the nested multiplex PCR approach provides an important tool for future studies of AM fungi population genetics and evolution.     

Detection of Ocular Toxoplasma gondii Infection in Chronic Irregular Recurrent Uveitis by PCR

Toxoplasma gondii is a zoonotic parasite resulting in human infections and one of the infectious pathogens leading to uveitis and retinochoroiditis. The present study was performed to assess T. gondii infection in 20 ocular patients with chronic irregular recurrent uveitis (20 aqueous humor and 20 peripheral blood samples) using PCR. All samples were analyzed by nested PCR targeting a specific B1 gene of T. gondii. The PCR-positive rate was 25% (5/20), including 5% (1) in blood samples, 25% (5) in aqueous humor samples, and 5% (1) in both sample types. A molecular screening test for T. gondii infection in ocular patients with common clinical findings of an unclear retinal margin and an inflammatory membrane over the retina, as seen by fundus examination, may be helpful for early diagnosis and treatment.