Decreased corneal sensation as an initial feature of Acanthamoeba keratitis

BACKGROUND: Herpes simplex keratitis is the most common misdiagnosis in patients with Acanthamoeba keratitis, which is increasing in frequency and is associated with daily wear soft contact lenses. Both entities usually present as unilateral keratitis. The manifestations of superficial Acanthamoeba keratitis (i.e., unilaterality, dendriform appearance, positive response to antivirals, and decreased corneal sensation) increase the opportunity for misdiagnosis as herpes simplex keratitis. The authors have encountered six patients with Acanthamoeba keratitis in whom the correct diagnosis was delayed from 2 weeks to 3 months. METHODS: All six patients underwent testing with the Cochet-Bonnet esthesiometer and extensive pharmacologic treatment for herpes simplex keratitis. Corneal scrapings were taken between 2 and 6 weeks after the initial examination. RESULTS: In all six patients, corneal sensation was decreased significantly. Drug therapy was ineffective. Cultures were positive for Acanthamoeba. Five of six patients underwent penetrating keratoplasty. CONCLUSIONS: Decreased corneal sensation has contributed to the misdiagnosis of Acanthamoeba as herpes simplex keratitis. Misdiagnosis results in delayed treatment and worse outcome. The authors found that significantly decreased corneal sensation is a frequent finding in early Acanthamoeba keratitis. Therefore, physicians should consider Acanthamoeba keratitis as an alternative diagnosis in patients with presumed herpes simplex keratitis with decreased corneal sensation.     

Vascular permeability factor/vascular endothelial growth factor and its receptors in oral and laryngeal squamous cell carcinoma and dysplasia

BACKGROUND: Herpetic keratitis is the main infectious cause of corneal opacity. The existence of effective antiviral agents underscores the need of an early diagnosis. AIM: To correlate clinical features of herpetic keratitis with virological studies. PATIENTS AND METHODS: Forty one patients with a clinical diagnosis of herpetic keratitis were studied. Viral isolation, polymerase chain reaction (PCR) and typification were done in a sample taken by swabbing the ocular lesion. RESULTS: Twenty six patients (31% female) had epithelial keratitis, that was mild or moderate in 88% of cases and acute in 77% of them. In 20 patients (77%), viral isolation and PCR were positive (HSV-2 in one case). Fifteen patients (67% female) had stromal keratitis, 93% of cases were moderate or severe and 53% were acute. Viral isolation was negative in all cases and in 20% PCR was positive. CONCLUSIONS: Viral isolation and PCR were equally sensitive in epithelial keratitis, but in stromal keratitis only PCR could detect the virus. Moderate acute dendrite was the predominant clinical manifestation. The higher proportion of women with stromal keratitis supports its possibly autoimmune etiology. HSV-2 is seldomly isolated and possibly associated to vertical transmission.     

Comparison of PCR assay with bacterial culture for detecting Streptococcus pneumoniae in middle ear fluid of children with acute otitis media

We have studied etiological diagnosis of acute otitis media (AOM) by comparing a newly developed pneumococcal PCR for Streptococcus pneumoniae to bacterial culture with 180 middle ear fluid (MEF) samples of 125 children with 125 episodes of AOM. For pneumococcal PCR assay, DNA from MEF samples was extracted by phenol-chloroform. The outer primers used amplified a 348-bp region of the pneumolysin gene, and the inner primers amplified a 208-bp region. S. pneumoniae was cultured in 33 (18%) samples, and pneumolysin PCR was positive for 51 (28%) of 180 MEF samples. Only 2 of 21 PCR-positive, S. pneumoniae culture-negative samples were positive for other otitis pathogens. By combining MEF culture and PCR results, 54 (30%) of 180 MEF samples had evidence of pneumococcal etiology. In conclusion, pneumolysin PCR is a sensitive and specific new method to study pneumococcal involvement in MEF samples of children with AOM.     

Sequence capture-PCR improves detection of mycobacterial DNA in clinical specimens

The rapid identification of mycobacterial DNA in clinical samples by PCR can be useful in the diagnosis of tuberculous infections, but several large studies have found that the sensitivity of this approach is not better than that of culture. In order to improve the sensitivity of detection of mycobacterial DNA in clinical specimens from patients with paucibacillary forms of tuberculosis, we have developed a procedure permitting the specific capture of mycobacterial DNA in crude samples prior to amplification, thereby concentrating the target sequences and removing irrelevant DNA and other potential inhibitors of the amplification reaction (sequence capture-PCR). By using this approach to capture and amplify two different sequences specific for organisms of the Mycobacterium tuberculosis complex (IS6110 and the direct repeat region), it was possible to detect as little as one genome of mycobacterial DNA in samples containing up to 750 micrograms of total DNA, representing a 10- to 100-fold increase in sensitivity compared with that obtained by purifying total DNA prior to amplification. Detection of the IS6110 sequence in pleural fluid samples from patients with tuberculous pleurisy by sequence capture-PCR gave positive results in 13 of 17 cases, including 3 of 3 culture-positive samples and 10 of 14 culture-negative samples. In contrast, when total DNA was purified from these samples by adsorption to a silica matrix prior to amplification, only the three culture-positive samples were positive by PCR. The sensitivity of detection of the direct repeat sequence in these samples by sequence capture-PCR was similar to that of IS6110 and, in addition, permitted immediate typing of the strains from some patients. We conclude that sequence capture-PCR improves the sensitivity of detection of mycobacterial DNA in paucibacillary samples. This approach should be useful in detecting rare target sequences from organisms implicated in other pathologic processes.     

Evaluation of a novel PCR-based diagnostic assay for detection of Mycobacterium tuberculosis in sputum samples

We report on a PCR-based assay we have developed for the detection of Mycobacterium tuberculosis in sputum samples. One hundred sputum specimens, which included 34 culture-positive and 66 culture-negative specimens, were evaluated with this system. Of the 34 culture-positive specimens, 31 were PCR positive, and 60 of the culture-negative specimens were PCR negative. An internal standard has been included in the assay system to monitor PCR inhibition and to confirm the reliability of the PCR assay.     

Early clinical diagnosis of acanthamoeba keratitis. A study of 70 eyes

BACKGROUND: Acanthamoeba keratitis is an uncommon condition which is usually associated with contact lens wear. The use of home made saline and poor hygiene are important risk factors. Early diagnosis is crucial since these cases respond well to medical therapy. The purpose of this paper is to describe and demonstrate early clinical signs. METHOD: Between September 1992 and October 1994, 70 cases of acanthamoeba keratitis, one of them bilateral, were prospectively monitored at Moorfields Eye Hospital in London. A database of all patients was set up and the clinical findings, diagnostic methods, therapeutic interventions and the outcome were recorded. RESULTS: 66 patients (96%) were contact lens wearers, 64 of them (97%) wore soft lenses. The mean interval between first symptoms and correct diagnosis was 42%. The most frequent initial diagnoses were "unclear keratoconjunctivitis" and "herpetic keratitis". Early corneal findings included punctate keratopathy (n = 14; 20%), pseudodendrites (n = 4; 6%), epithelial infiltrates (n = 17; 24%), diffuse or focal sub-epithelial infiltrates (n = 36; 51%) and radial keratoneuritis (n = 5; 7%). Ring infiltrates (n = 13; 18%) and corneal ulceration (n = 13) were late signs. CONCLUSION: When the above corneal findings are observed, particularly in contact lens wearers, the diagnosis of acanthamoeba keratitis should be considered. The diagnosis of "herpetic keratitis" in association with contact lens wear should be encountered with scepticism.     

EMLA cream provides rapid pain relief for the curettage of molluscum contagiosum in children with atopic dermatitis without causing serious application-site reactions

BACKGROUND: Determination of the etiology of pneumonia in young children is difficult because blood culture, the usual method of diagnosis, is positive in only a small proportion of cases. For this reason vaccine trials that include bacterial pneumonia as an endpoint must be large. OBJECTIVES: To determine whether a diagnostic test based on a polymerase chain reaction could be used as an alternative to conventional blood culture for diagnosis of invasive Haemophilus influenzae type b (Hib) infections in young children investigated during the course of a large vaccine trial. METHODS: DNA was extracted from blood culture supernatants and probed for the presence of Hib DNA with a PCR assay with primers derived from the cap gene locus of Hib. Results of the PCR assay were compared with those obtained by conventional culture techniques. RESULTS: Blood cultures were obtained from 1544 children with suspected pneumonia, meningitis or septicemia and from 31 healthy control children who were contacts of cases. Blood culture supernatants were tested for Hib DNA in the PCR test. The sensitivity and specificity of a positive PCR test in blood culture supernatant as against culture of Hib from any normally sterile site were 100 and 99%, respectively. Eleven children had positive Hib PCR tests on blood culture supernatants but were negative by culture. In one of these cases Hib was isolated from a lung aspirate and in two other patients H. influenzae strains other than Hib were obtained from the cerebrospinal fluid. Eight of these 11 children were in the control group. When the results of the PCR assay were used to determine vaccine efficacy, a value of 86% was obtained compared with a figure of 95% obtained when conventional culture techniques were used. CONCLUSIONS: An Hib PCR assay on blood culture supernatants proved to be sensitive and specific for the diagnosis of Hib disease in children. The distribution of PCR-positive, culture-negative cases between Hib-vaccinated and control groups paralleled that of culture-positive cases, suggesting that most of these children had been infected with Hib. A trial of a highly efficacious vaccine provides a novel way for evaluating new diagnostic tests for which there is no standard diagnostic test of 100% reliability.     

Diagnosis of pulmonary tuberculosis by the amplicor test for Mycobacterium tuberculosis in induced sputum

The polymerase chain reaction (PCR) technique was compared with the conventional smear and culture method for the detection and identification of Mycobacterium tuberculosis in 25 induced sputa samples. Sputum induction was performed with an ultrasonic nebulizer using 3% hypertonic saline in 27 previously untreated patients suspected of active pulmonary tuberculosis clue to chest X-ray findings, but who were unable to spontaneously expectorate sputum. All patients received anti-tuberculosis drugs for at least 6 months after sputum induction. Two patients could not expectorate sputum after hypertonic saline inhalation. Microscopy failed to detect positive acid-fast bacilli in 25 induced sputa samples. Induced sputa were both PCR- and culture-positive for 10 patients, PCR-positive and culture-negative for 4 patients, and PCR-negative and culture-positive for 2 patients. These results suggest that the PCR method is useful in diagnosing tuberculosis in induced sputum, and that the results of PCR and culture Procedures can complement each other.     

Rapid diagnosis of tuberculous meningitis by polymerase chain reaction assay of cerebrospinal fluid

A polymerase chain reaction (PCR) method for the rapid diagnosis of tuberculous meningitis (TBM) was used to study prospectively 47 cerebrospinal fluid (CSF) samples from 45 patients. Twenty CSF samples were from patients with clinically suspected TBM and another 27 samples came from patients without clinically suspected TBM. Mycobacterial DNA was detected in 15 CSF samples (14 from patients with clinically suspected TBM and 1 from a patient not suspected of having TBM). Of the PCR-positive samples, 4 were also positive for mycobacterial culture. However, 32 PCR-negative samples were all culture-negative. All samples were negative for the acid-fast bacillus by direct smear. The single PCR-positive patient in the clinically unsuspected TBM group was initially diagnosed as suffering from aseptic meningitis on the basis of his clinical features. The mycobacterial culture of his CSF specimen was also positive and a revised diagnosis of an aseptic type of TBM was made. The estimations of specificity and sensitivity in this study were 100% and 70% respectively. The results showed that using a PCR to detect mycobacterial DNA in CSF for the early diagnosis of TBM is not only a rapid but also an accurate method.     

Evaluation of PCR in detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues: comparison of four amplification assays

We compared the sensitivities and specificities of four nested PCR assays for the detection of Mycobacterium tuberculosis from formalin-fixed, paraffin-embedded tissues. Thirty-seven autopsy samples from human immunodeficiency virus-positive patients were analyzed: 15 were M. tuberculosis positive, 11 served as negative controls, and 11 were Ziehl-Neelsen positive without cultural confirmation of M. tuberculosis. Three genomic sequences (mtp40, 65-kDa antigen gene, and IS6110) with different molecular masses and numbers of repetitions within the M. tuberculosis genome were targeted. On the IS6110 sequence, two fragments of different sizes (106 and 123 bp, respectively) were amplified with two separate pairs of primers. The highest sensitivity rates were obtained by amplifying the highly repetitive IS6110 insertion sequence, and the different primers tested showed a sensitivity ranging from 80 to 87%. Amplification of the large 223-bp fragment of the mtp40 sequence present in a single copy in the M. tuberculosis genome yielded a high rate of false-negative results, ranging from 66 to 80%. A poor sensitivity (from 47 to 60%) was also shown by PCR amplification of the 142-bp 65-kDa antigen gene. All the PCRs except that for the 65-kDa antigen gene showed a specificity of 100%. Moreover, different results were obtained with different dilutions of DNA, and DNA concentrations of 1 and 3 microg yielded the highest sensitivities depending upon which protocol was used. Application of the PCRs to the Ziehl-Neelsen-positive, culture-negative samples confirmed the sensitivities of the PCRs obtained with the control samples. In conclusion, PCR can successfully be used to detect M. tuberculosis from paraffin-embedded tissues and can be particularly useful in the validation of a diagnosis of tuberculosis in clinical settings in which the diagnosis is uncertain. However, the efficacy of PCR strictly depends on several amplification parameters such as DNA concentration, target DNA size, and the repetitiveness of the amplified sequence.     

Acanthamoeba keratitis with symbiosis of Hartmannella ameba

PURPOSE: To report a case of severe amebic keratitis in which both Hartmannella and Acanthamoeba were isolated simultaneously from the same lesion. METHOD: Case report. The deep corneal lesion was scraped for cytopathology and isolation of the pathogens. We tested the in vitro sensitivities of the pathogens to several drugs. RESULTS: Cultures of the corneal scrapings and of the solution in the patient's contact lens storage case were positive for Acanthamoeba E9 cysts and trophozoites. Hartmannella ameba coexisted with Acanthamoeba in the cornea. When tested in vitro, Acanthamoeba trophozoites were sensitive to both miconazole nitrate and natamycin, while cysts were sensitive only to natamycin. However, the patient did not respond to these antiamebic drugs. CONCLUSIONS: This case suggests that Acanthamoeba is not the only origin of amebic keratitis. Hartmannella may also cause severe drug-resistant keratitis.     

The diagnostic significance of polymerase chain reaction for ocular samples in viral retinitis

During the past two years we studied the incidence of infection by eight members of the herpesvirus family in ocular samples (tear fluid, the aqueous, and the vitreous) using the polymerase chain reaction (PCR). A total of 31 ocular samples were collected from 22 eyes of 13 patients. The series comprised five cases of acute retinal necrosis, four of cytomegalovirus retinitis, three of proliferative diabetic retinopathy, and one of ocular malignant lymphoma. In 12 eyes of 9 patients with viral retinitis, causative viral DNA was detected either from the tear fluid (1/12, 8%), the aqueous (6/7, 86%), or the vitreous (1/1, 100%). In the fellow eyes free of viral retinitis, no viral DNA was detected in the samples (6 of tear fluid and one of the aqueous). Out of the other 4 patients without viral retinitis, viral DNA was detected in one patient with ocular malignant lymphoma. The findings show that PCR is a useful and sensitive method in the diagnosis of viral retinitis, but that it may give false positive results. The aqueous and the vitreous samples gave more positive results than tear fluid.     

Evaluation of effective treatment drugs against Acanthamoeba cyst

Cysts of 2 isolates of Acanthamoeba from the cornea of 2 patients with confirmed Acanthamoeba keratitis were tested in vitro for sensitivity to antimycotic agents such as fluconazole, miconazole, amphotericin-B, pimaricin, antiprotozoal agents such as pentamidine isetionate and antiseptics which could be use in the ophthamological region. Pimaricin was the most successful cysticidal agent against the two strains. Sensitivity to pentamidine isetionate showed variation. Fluconazole, miconazole and amphotericin-B were resistant against cysts with concentration of eye drops that have been used in the treatment of Acanthamoeba keratitis. It was supposed that 5% pimaricin eye drops could be use in the treatment of Acanthamoeba keratitis in addition to keratomycosis. Pentamidine isetionate which belong to the diamidine family, is not yet clear as to the side effects to corneal epithelium cell, but we believe that this drug could be expected as a new therapeutic agent for Acanthamoeba keratitis.     

Amebic keratitis in a wearer of disposable contact lenses due to a mixed Vahlkampfia and Hartmannella infection

PURPOSE: To support the hypothesis that Acanthamoeba is not a unique cause of amebic keratitis, we report a case of amebic keratitis in which viable Acanthamoeba could not be isolated from corneal tissue. Vahlkampfia and Hartmannella, two other genera of free-living ameba, were isolated, however, using prolonged culture. METHODS: A 24-year-old wearer of soft contact lenses had keratitis. Extensive histologic and microbiologic investigations were performed on corneal scrape, biopsy, and keratoplasty tissue. Contact lenses, storage case, and the home water supply, where contact lens hygiene was practiced, were examined for the presence of micro-organisms. RESULTS: No viruses, pathogenic bacteria, or fungi were detected from corneal tissue samples. Amebae were observed using light and electron microscopy, but these could not be unequivocally classified using immunocytochemical staining. Viable Vahlkampfia and Hartmannella, but no Acanthamoeba, were isolated from the corneal biopsy sample. Indirect immunofluorescence with a range of polyclonal rabbit antisera raised against axenically cultivated stains of the three amebal genera was unhelpful because of cross-reactivity. A diverse range of micro-organisms was present within the storage case, including the three amebal species. Amebic cysts also were associated with the contact lens. CONCLUSION: A mixed non-Acanthamoeba amebic keratitis has been identified in a wearer of soft contact lenses where lack of storage case hygiene provided the opportunity for the free-living protozoa Vahlkampfia and Hartmannella to be introduced to the ocular surface. When Acanthamoeba-like keratitis occurs, but where Acanthamoeba cannot be isolated using conventional laboratory culture methods, alternate means should be used to identify other amebae that may be present. Polyclonal immunofluorescent antibody staining was unreliable for generic identification of pathogenic free-living amebae in corneal tissue.     

Value of polymerase chain reaction (PCR) for diagnosis of tuberculoid meningitis

The prognosis of tuberculous meningitis (TBM) depends on early therapy based on rapid diagnosis. To study the clinical value of PCR in diagnosis of TBM, we investigated CSF specimens from 49 patients. After cell lysis and DNA preparation following a standard protocol, we performed a half-nested PCR with primers able to detect mycobacterial DNA. PCR results were evaluated according to clinical features, histopathological data, and bacteriological results. PCR detected four of five cases of confirmed TBM, corresponding to a sensitivity of 80%. Positive PCRs were also obtained in 25% CSF samples of non-TBM patients. Most of these false positive results were due to amplification of Mycobacteria fortuitum (M. fortuitum) as determined by direct sequencing analysis. To enhance specificity of our half nested protocol, the oligonucleotide primers that were specific for several mycobacterial subspecies were substituted by a primerpair, which allows selective amplification of DNA from Mycobacteria tuberculosis (M. tuberculosis). By using the altered PCR protocol, the screening of CSF samples revealed a much higher specificity (97%) and constant sensitivity (80%) in diagnosis of TBM. These findings indicate, that M. fortuitum, as an ubiquitous mycobacterial subtype of low pathogenicity, can potentially contaminate clinical specimens and account for false positive PCR results. Therefore, the clinical value of PCR in diagnosis of TBM strongly depends on appropriate oligonucleotide primers, that allow to differentiate between mycobacterial subtypes.     

Comparison of sample preparation methods for detection of Legionella pneumophila in culture-positive bronchoalveolar lavage fluids by PCR

A prospective study was conducted on 25 Legionella pneumophila culture-positive and 98 culture-negative bronchoalveolar lavage fluid samples to compare two DNA preparation methods: a rapid modified Chelex-based protocol and a proteinase K method. PCR was found to be more sensitive with the Chelex-based method (P = 0.03). N difference was found concerning the inhibition rate.     

Detection of vancomycin-resistant enterococci in fecal samples by PCR

Surveillance cultures for vancomycin-resistant enterococci (VRE) are time-consuming and expensive for the laboratory to perform. Therefore, we investigated the use of PCR as an alternative method of detecting and identifying VRE directly in fecal samples. PCR primers directed to vanA, vanB, vanC1, vanC2, and enterococcal ligase genes were used to detect and identify VRE in fecal material obtained by rectal or perirectal swabbing. Although PCR-inhibitory substances were present in DNA prepared directly from the swabs, the inhibitory substances could be reduced by processing the nucleic acid with two commercially available DNA preparation columns. Fecal material from 333 swabs was cultured on several selective agar media before and after broth enrichment. DNA was extracted from the fecal material and was analyzed by PCR. By using all four primer sets, only 59 (67.8%) of the samples were positive for vanA. However, after retesting the negative samples with only the vanA primer set, 77 (88.5%) of 87 specimens that were culture positive for Enterococcus faecium containing vanA were positive by PCR. One specimen was PCR positive for the vanA gene but culture negative for enterococci. The specificity of the vanA assay was 99.6%. PCR analysis of enrichment broth samples with all four primers sets after 15 to 18 h of incubation detected 74 (85.1%) of the 87 culture-positive specimens. The specificity of the vanA assay after the enrichment step was 100%. No vanB-containing enterococci were recovered by culture. Since 16 samples can be tested by PCR in 4 h (including electrophoresis), identification of VRE is possible within 8 h of specimen submission at a cost of approximately $10.12/assay. Thus, PCR may be a cost-effective alternative to culture for surveillance of VRE in some hospitals.     

Rapid diagnosis of Mycobacterium tuberculosis bacteremia by PCR

A method based on DNA amplification and hybridization has been used for the rapid detection of Mycobacterium tuberculosis in blood samples from 38 hospitalized patients (15 human immunodeficiency virus [HIV] positive and 23 HIV negative) in whom localized or disseminated forms of tuberculosis were suspected. In 32 of these patients, the diagnosis of tuberculosis was eventually confirmed by conventional bacteriological or histological procedures. M. tuberculosis DNA was detected with the PCR technique in the peripheral blood mononuclear cells from 9 of 11 (82%) HIV-infected patients and in 7 of 21 (33%) HIV-negative patients (P < 0.01), while M. tuberculosis blood cultures were positive in 1 of 8 (12.5%) and 1 of 18 (5.5%) patients, respectively. PCR was positive in all cases with disseminated disease in both HIV-negative and HIV-positive patients and also in the HIV-positive patients with extrapulmonary tuberculosis. Seven samples from patients with documented illness other than tuberculosis and 12 specimens from healthy volunteers, including seven volunteers with a recent positive purified protein derivative test, were used as controls and had a negative PCR. These results suggest that detection of M. tuberculosis DNA in peripheral blood mononuclear cells may be a useful tool for rapid diagnosis of disseminated and extrapulmonary forms of tuberculosis, especially in an HIV-positive population.     

Neuroradiology case of the day. Burkitt lymphoma

We describe the clinical and laboratory manifestations of human granulocytic ehrlichiosis (HGE) in eight patients for whom cultures were positive for the HGE agent and compare them with 15 patients for whom cultures were negative but who fulfilled a modified New York State Surveillance definition for HGE. Polymerase chain reaction analysis was positive in 8 (100%) of 8 culture-positive cases vs. 3 (20%) of 15 culture-negative cases (P < .001), morulae were detected in 7 (100%) of 7 culture-positive cases in which tests were performed vs. 0 of 15 culture-negative cases (P < .001), and a fourfold change in antibody titer was demonstrated in 6 (75%) of 8 culture-positive cases vs. 9 (69%) of 13 culture-negative cases (P = not significant). Patients for whom cultures were positive had higher mean oral temperatures +/- SD at presentation than did patients for whom cultures were negative (38.6 degrees C +/- 0.7 degree C vs. 37.2 degrees C +/- 0.8 degree C, respectively; P = .002). Other symptoms and signs were not significantly different between the two groups. Multivariate analysis revealed that the lymphocyte count at presentation was significantly lower in culture-positive cases than in culture-negative cases. Clinical response to treatment was similar in the two groups. Culture confirmation of HGE is the gold standard for defining the sensitivity and specificity of other diagnostic tests presently being developed.