Free-living amoebae as opportunistic and non-opportunistic pathogens of humans and animals.

Infections caused by Naegleria fowleri, Acanthamoeba spp., and Balamuthia mandrillaris occur throughout the world and pose many diagnostic challenges. To date, at least 440 cases of severe central nervous system infections caused by these amebas have been documented worldwide. Rapid and specific identification of these free-living amebas in clinical samples is of crucial importance for efficient case management. We have developed a triplex real-time TaqMan PCR assay that can simultaneously identify Acanthamoeba spp., B. mandrillaris, and N. fowleri in the same PCR vessel. The assay was validated with 22 well-characterized amebic strains harvested from cultures and nine clinical specimens that were previously characterized by in vitro culture and/or immunofluorescence assay. The triplex assay demonstrated high specificity and a rapid test completion time of less than 5 h from the reception of the specimen in the laboratory. This assay was able to detect one single ameba per sample analyzed, as determined with cerebrospinal fluid spiked with diluted cultured amebas. This assay could become useful for fast laboratory diagnostic assessment of amebic infections (caused by free-living amebas) in laboratories with adequate infrastructure to perform real-time PCR testing.

Multiplex Real-Time PCR Assay for Simultaneous Detection of Acanthamoeba spp., Balamuthia mandrillaris, and Naegleria fowleri

Healthy individuals are known to shed herpesviruses. Humans are reservoirs, and asymptomatic shedding is a major factor in the spread of the virus. In a recent 14-month study, Epstein-Barr virus (EBV) was present in all 30 individuals tested, and 4 (13%) of 30 shed cytomegalovirus (CMV) in urine.1 In a cross-sectional study in Eritrea, Africa, with the exception of children 80% herpes simplex virus type 1 (HSV-1) seropositive, and >90% CMV and varicella zoster virus (VZV) seropositive.2 Antibodies for HSV-1 have also been detected in tears (73%) and saliva (2.5%) of 40 healthy individuals.3

HSV-1 and, to a lesser extent, HSV-2 are known to be the leading causes of virus-induced blindness in the Western world, with approximately 500,000 individuals having herpetic eye disease in the United States.4 More than 62% of the U.S. population >12 years of age is positive for HSV-1, HSV-2, or both.4 Worldwide, 60% to 90% of the adult population is HSV-1 antibody positive.5,6 In one study, 100% of individuals older than 60 years were found to be HSV-1 seropositive.7 Despite the prevalence of HSV infections, however, only a small number of latently infected humans experience symptomatic disease. Only 1% to 6% of primary infections are clinically recognized.8 Consequently, asymptomatic shedding of HSV is considered the major form of transmission.

Thus far, studies have focused on quantifying HSV-1 and -2 antibody titers and/or frequency of shedding in patients with active herpesvirus lesions or immediately after the time of active lesions.9 Although HSV-1 seropositivity in healthy individuals has also been evaluated,3,10 asymptomatic shedding of HSV-1, especially in tears, has received less attention. To the best of our knowledge, only three studies have evaluated HSV-1 shedding in tears and saliva of healthy individuals, using relatively insensitive culturing techniques.11–13

Detection of HSV shedding is dependent on the population surveyed, as well as on the diagnostic technique used. Before the advent of real-time PCR, the presence of HSV could be determined only by relatively insensitive culturing of infectious virus.14–21 Today we are able to detect HSV DNA with PCR, the new gold standard for HSV detection in clinical samples. Hence, the percentage of individuals detected as positive for HSV-1 DNA is expected to increase significantly, as has the percentage of individuals shedding EBV in saliva.1

To the best of our knowledge, the present study is the largest cross-sectional assessment performed to date of the presence of HSV-1 DNA in the eyes and mouths of healthy individuals, in terms of population size and total samples collected. We used real-time PCR to quantify the presence and frequency of asymptomatic viral shedding in a combined total of 5529 tear and saliva specimens. Samples consisted of mouth and eye swabs collected twice daily from 50 volunteers over the course of 30 days. The subjects were instructed to swab the mouth and one eye morning and night. In addition, serum HSV IgG antibody titers were determined. We found that 98% (49/50) of the subjects in our study were positive at least once for HSV-1 DNA in tears and/or saliva.

HSV-1 DNA in tears and saliva of normal adults

The persistent dilemma of microbial keratitis: Global burden, diagnosis, and antimicrobial resistance.

https://www.cell.com/trends/parasitology/pdf/S1471-4922(06)00048-1.pdf

The pathophysiology of Acanthamoeba keratitis.

Identification of Acanthamoeba cysts and trophozoites in ocular tissues requires considerable expertise and is often time-consuming. An 18S rRNA gene-based PCR test, highly specific for the genus Acanthamoeba, has recently been reported in the molecular diagnosis of Acanthamoeba keratitis. This PCR assay was compared with conventional microbiological tests for the diagnosis of Acanthamoeba keratitis. In a pilot study, the PCR conditions with modifications were first tested on corneal scrapings from patients with culture-proven non-contact lens-related Acanthamoeba, bacterial, and fungal keratitis. This was followed by testing of corneal scrapings from 53 consecutive cases of microbial keratitis to determine sensitivity, specificity, and predictive values of the assay. All corneal scrapings from patients with proven Acanthamoeba keratitis showed a 463-bp amplicon, while no amplicon was obtained from patients with bacterial or fungal keratitis. Some of these amplified products were sequenced and compared with EMBL database reference sequences to validate these to be of Acanthamoeba origin. Out of 53 consecutive cases of microbial keratitis included for evaluating the PCR, 10 (18.9%) cases were diagnosed as Acanthamoeba keratitis on the basis of combined results of culture, smear, and PCR of corneal scrapings. Based on culture results as the “gold standard,” the sensitivity of PCR was the same as that of the smear (87.5%); however, the specificity and the positive and negative predictive values of PCR were marginally higher than the smear examination (97.8 versus 95.6%, 87.5 versus 77.8%, and 97.8 versus 97.7%) although the difference was not significant. This study confirms the efficacy of the PCR assay and is the first study to evaluate a PCR-based assay against conventional methods of diagnosis in a clinical setting.

Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis in Non-Contact Lens Wearers in India

An insight into the PB1F2 protein and its multifunctional role in enhancing the pathogenicity of the influenza A viruses.

Objectives To establish that the protozoan Acanthamoeba isone of the causative organisms associated with non–contact lens–relatedkeratitis in the Indian population and to develop a simple and sensitive diagnosticassay for clinical testing. Design DNA sequencing of nuclear 18S and 26S ribosomal DNA motifs was performedand compared with the reference Acanthamoeba strains,to establish the genetic identity of the putative amoeba isolates obtainedfrom the corneal scrapings of non–contact lens–wearing patientswith keratitis. Ribosomal DNA typing of clinical corneal scrapings from thepatients with keratitis was performed by means of a simple agarose gel–basedmultiplex polymerase chain reaction assay, to detect the cases of Acanthamoeba keratitis. Results The ribosomal DNA analysis of 15 putative amoeba isolates obtained fromthe corneal scrapings of 14 patients with keratitis and 1 from the patients'environment established the isolates to be pathogenic formsof Acanthamoeba belonging to type T4 ribosomal DNA genotype. Multiplexpolymerase chain reaction assay was specific and sensitive enough to detectas low as 5 pg of Acanthamoeba DNA. Its utility asa reliable diagnostic assay was demonstrated directly with the use of 34 additionalcorneal scrapings. Conclusions Acanthamoeba is one of the causative organismsof keratitis in Indian patients with no history of contact lens usage. Moreover,the Acanthamoeba infection can be easily detectedin the clinical samples by means of the simple multiplex polymerase chainreaction assay based on ribosomal DNA typing. Clinical Relevance This study suggests the need and means to determine the incidence andprevalance of Acanthamoeba keratitis in India andelsewhere. Moreover, the polymerase chain reaction assay would help in earlyand definitive diagnosis, leading to better prognosis of Acanthamoeba keratitis condition.

https://jamanetwork.com/journals/jamaophthalmology/articlepdf/416600/ECS30045.pdf

Acanthamoeba keratitis in non-contact lens wearers in India: DNA typing-based validation and a simple detection assay

Purpose. A majority of ocular viral diseases are caused by herpes group of viruses. Such infections, especially atypical herpetic keratitis, iridocyclitis and intra-ocular inflammations, can often present with overlapping clinical manifestations misleading the diagnosis. Molecular techniques are most useful in such instances for an accurate and rapid diagnosis since conventional methods are time consuming and less sensitive. A multiplex PCR was developed and used for the detection of herpes simplex virus (HSV), varicella zoster virus (VZV), and cytomegalovirus (CMV) in ocular samples. Methods. One hundred and forty six ocular samples (corneal scrapings – 52, aqueous fluid – 36, vitreous fluid – 31, tissues – 26, skin vesicle scraping – 1) were included in the study. The sensitivity of the assay was determined using serial dilutions of standard strains of HSV, VZV, and CMV vis-a-vis plaque forming assay. Results. The sensitivity of the assay was 4, 4 and 12PFU/ml or 20, 20 and 60 genome copy numbers of HSV...

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens

Results: The ribosomal DNA analysis of 15 putative amoeba isolates obtained from the corneal scrapings of 14 patients with keratitis and 1 from the patients’ environment established the isolates to be pathogenic forms of Acanthamoeba belonging to type T4 ribosomal DNA genotype.Multiplexpolymerasechainreactionassaywas specific and sensitive enough to detect as low as 5 pg of AcanthamoebaDNA. Its utility as a reliable diagnostic assay was demonstrated directly with the use of 34 additional corneal scrapings. Conclusions: Acanthamoeba is one of the causative organisms of keratitis in Indian patients with no history of contact lens usage. Moreover, the Acanthamoeba infectioncanbeeasilydetectedintheclinicalsamplesbymeans of the simple multiplex polymerase chain reaction assay based on ribosomal DNA typing. Clinical Relevance: This study suggests the need and meanstodeterminetheincidenceandprevalanceofAcanthamoebakeratitis in India and elsewhere. Moreover, the polymerase chain reaction assay would help in early and definitive diagnosis, leading to better prognosis ofAcanthamoeba keratitis condition.

Gunisha Pasricha

Papers

Use of 18S rRNA Gene-Based PCR Assay for Diagnosis of Acanthamoeba Keratitis in Non-Contact Lens Wearers in India

An insight into the PB1F2 protein and its multifunctional role in enhancing the pathogenicity of the influenza A viruses.

Acanthamoeba keratitis in non-contact lens wearers in India: DNA typing-based validation and a simple detection assay

Multiplex polymerase chain reaction for the detection of herpes simplex virus, varicella-zoster virus and cytomegalovirus in ocular specimens

Acanthamoeba Keratitis in Non-Contact Lens Wearers in India