increment of HSCT recipients [6, 7]. HSCT is a well established treatment for many hematological diseases such as acute leukaemia, myelodysplastic syndromes and aplastic anemia. The donor for HSCT was initially lim- ited to an identical twin, and then extended to human leucocyte antigen (HLA)-matched related or unrelated donor. Later, HSCT from a HLA-mismatched donor was also used in some patients lacking suitable HLA- matched donors . The rapid progress of HSCT has significantly lowered the rates of transplant-related mor- bidity and mortality. However, the broadening of indica- tion and the increase of transplant survivors after HSCT unfavorably increases many opportunistic infections in- cluding CMV retinitis [14, 15]. Here, we retrospectively described the clinical features of 12 CMV retinitis pa- tients following allogeneic HSCT. The purpose of this study was to analyze the pattern of this disease and summarize the potential factors affecting visual progno- sis in this specific population.
CMV viral load was quantified with the real-time PCR assay to explore whether the level of CMV viral load in blood was correlated to the severity of CMV retinitis in this study. There was limitation to detect the level of CMV viral load in blood, sometimes CMV in the blood does not rule out the possibility of a different infection in the eye. It was reported that the CMV-PCR assay had a high predicting value with estimated sensitivity of 95% in detecting untreated CMV retinitis if vitreous samples were used. However, the vitreous sampling proce- dure has a risk to cause hemorrhage, infection and vision acuity loss, and was difficult to obtain patient’s consent. So vitreous sampling is reserved for unusual cases with atypical features, where clinical findings are ambiguous and there needs to be clarification between other retinitis.
Study design. The original study consisted of 725 participants ran- domized in a 2:1 ratio to receive ganciclovir or placebo, respectively. The 619 (85%) patients evaluated in this viral load study comprised all persons for whom baseline plasma samples were available (201 placebo recipients and 418 ganciclovir recipients). The median CD4 1 lymphocyte count for the cohort was 21 cells per microliter, 99% were males and the median age was 38 yr. No study participants had a history of CMV disease and none had received CMV antiviral ther- apy. Participants had no evidence of CMV retinitis on dilated eye ex- amination by an experienced ophthalmologist before entry into the study. Participants were evaluated every 2 mo for signs consistent with clinical CMV disease and were examined by an experienced ophthalmologist for the presence of CMV retinitis. Full criteria for study entry and establishing the presence of CMV disease have been described in detail previously (10). The study was conducted before the availability of antiretrovirals with activity against the HIV pro- tease.
Patient population. HIV-seropositive patients were enrolled randomly from among the participants in various HIV treatment trials at the Center for AIDS Research at the Stanford University School of Medicine and its affiliated subunit at Kaiser Permanente Medical Center, San Francisco, Calif. CD4 cell counts for the patients in this study were quantitated by fluorescence-activated cell sorting analysis. CD4 cell counts were available for each patient within 30 days of sample acquisition. Forty-nine patients with CD4 counts of , 100 (mean 6 standard deviation 5 32 6 27) were free of any known CMV disease. CMV retinitis (n 5 22) was diagnosed ophthalmologically by the typical clinical appearance of ret- inal hemorrhage and exudate. CD4 cell counts in patients with retinitis were 39 6 25 (mean 6 standard deviation). CMV gastrointestinal disease in the symp- tomatic disease group (n 5 1) was diagnosed by both sigmoidoscopy and the presence of typical intranuclear and intracytoplasmic inclusions in stained tissue samples obtained by biopsy of the intestinal wall.
32.03% of the cases had posterior segment lesions in our study, HIV retinopathy (23.3%) being the most com- mon followed by CMV retinitis (3.9%). Similar trend was seen in the study done by Awan et al.  in Africa. In a study by N. A. V. Beare et al.  in Malawi, Africa only 0.5% of the cases had signs of CMV retinitis. This may be the result of mortality early in the disease course, or differences in race, HIV subtype, or comorbidity. But this was different from other studies done in USA and India as shown in Table 5. The occurrence of CMV retinitis was 37% in the study by Jabs et al. , 17% by J. Biswas  and 10.65% by Sriprakash K. S. et al. . This may be because the study conducted in the USA was before the introduction of the HAART and in India [14,22] it was before the widespread use of HAART as mentioned in those studies. The occurrence of Retinal Detachment seen in our study was less than in other studies and this may be due to the use of HAART. In 2 cases (1.9%), the retinal degeneration with was seen. The retinal degeneration observed may be the sequelae of CMV retinitis. In our study we found one case having CSR in both eyes. Though not mentioned in any journals or textbooks as the posterior segment finding in HIV/ AIDS, it may be unrelated to HIV/AIDS and seen coin- cidentally.
to prove that oral valganciclovir was not 10% worse than intravenous ganciclovir. The study enrolled 160 patients with newly diagnosed CMV retinitis; 80 of them received induction therapy with intravenous ganciclovir 5 mg/kg for three weeks and the remaining 80 received induction therapy with oral valganciclovir 900 mg twice daily for three weeks. The patients were followed with the same therapy once daily for one week. All patients were then placed on maintenance treatment with oral valganciclovir 900 mg once daily. Retinal photographs were obtained at baseline visit and then week two and week four and evaluated by masked reviewers. Although most patients had zone-3 retinitis, 24% in each arm had zone-1 retinitis. The mean CD4 cell count at baseline was 54 cells/mm 3 in the intravenous ganciclovir
studies to identify mechanisms underlying this interaction have not been carried out in humans. Cross-sectional studies on changes in CMV-specific immunity in HIV infection have fo- cused chiefly on humoral and proliferative T-cell responses (8, 12, 37). Asymptomatic HIV-infected people with normal CD4 1 T-cell counts had decreased proliferative responses to specific CMV antigens (12). Decreased T-cell proliferative re- sponses to CMV in patients with AIDS was a predictor for developing CMV retinitis, particularly in those with HLA DR7, B44, or B51 haplotypes (37). The only published studies on CMV-specific CTL activity in HIV infection used an insen- sitive technique for CTL detection. Using fresh PBMC without in vitro stimulation, CTL activity was not detected in AIDS patients with CMV viremia but was detected in a proportion of renal transplant recipients with active CMV infection (35, 36). The rhesus model is ideally suited for in vivo prospective stud- ies, and the similarities between simian and human CMV, as well as SIV and HIV, support the validity of this experimental system as a model for the study of CMV pathogenesis in AIDS. Characterization of CMV-specific CTL responses in rhesus macaques and the ability to maintain CMV-seronegative ani- mals free of CMV infection have provided us with powerful tools for such studies.
Various literature gives incidence of CMV retinitis as high as 15-40%. But in this study group, CMV retinitis is reported in only 4% of total study group and 23.5% of those with posterior segment manifestations. The higher incidence in literature may be because the studies elsewhere conducted dealt with full blown cases of AIDS, while this study is a mixture of asymptomatic cases with fewer cases of AIDS.
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Huang et al evaluated the use of intravitreal ganciclovir injection as a loading dose with or without subsequent oral valganciclovir to treat CMV anterior uveitis. Two patients had only the initial intravitreal ganciclovir injection without oral valganciclovir and four patients had a mean of 2.3 months of oral valganciclovir after intravitreal treatment. The authors found that none of the patients had a recurrence of intraocular inflammation and all patients had significant improvement in visual acuity with significantly reduced IOP. Therefore, adding intravitreal ganciclovir might be a useful option in treating patients with CMV anterior uveitis. However, intravitreal ganciclovir frequently requires regular injections, and repeated intravitreal injections are associated with increased risks of intraocular infection, retinal break, and detachment. Because CMV anterior uveitis is less vision- threatening compared with CMV retinitis, we only used oral valganciclovir to treat patients with CMV anterior segment inflammation in order to avoid the potential risks associated with intravitreal ganciclovir injections.
C ongenital cytomegalovirus (cCMV) infection affects about 0.7% of newborns worldwide (1–3). The clinical CMV-re- lated sequelae at birth are highly variable and related to maternal serostatus and the time of onset of congenital infection during pregnancy (4–9). Whenever clinically evident, CMV-induced damages include sensorineural hearing loss (SNHL), visual im- pairment, delayed psychomotorial development, and retardation (10–13). Understanding the risk factors and biomarkers associ- ated with the maternal transmission of CMV infection represents a leading priority for both diagnosis and clinical management of cCMV. Recently, it was shown that maternal CMV cell-mediated immunity (CMI) plays a critical role in determining cCMV (14– 16). Several assays are available to assess CMV-specific CMI, and the large majority of these assays are based on interferon gamma (IFN-␥) release assays (IGRAs) (17–20). In this study, two IGRAs that detect CMV-specific CMI, the CMV enzyme-linked immu- nosorbent spot (ELISPOT) and CMV QuantiFERON assays, were compared for their prediction of cCMV. Both the CMV ELISPOT and CMV-QuantiFERON assays detect IFN- ␥ produced by anti- gen-stimulated peripheral blood mononuclear cells (PBMCs). The main differences between the assays include the antigen stim- ulus composition, with stimulation of CD8 ⫹ T-cell responses in the CMV QuantiFERON assay (21) and stimulation of both CD4 ⫹ and CD8 ⫹ T-cell responses in the CMV ELISPOT assay (22, 23). Moreover, the CMV QuantiFERON assay detects IFN-␥ in a volume of ⬃1 ml of whole blood, while the CMV ELISPOT assay detects IFN-␥ secreted by ⬃2 ⫻ 10 5 PBMCs (22, 23). Recent studies suggest that the CMV ELISPOT and CMV QuantiFERON assays may display large variability on an individual basis (24, 25).
To verify these parameters for downstream analysis, two internal controls were run. The first was a lim- ited passaged BAC clone for the CMV strain TR, which had an error rate of 0.03%, between two runs. The error rate was calculated by running the sample through the pipeline from the beginning and compar- ing the variants found at any location between the two runs divided by the number of possible locations, reported as a percentage [37–39]. Second, to confirm the reproducible nature of the pipeline from DNA isolation to variant calling, a patient sample was pre- pared and sequenced three times from the beginning of the pipeline resulting in an error rate of 0.09% of variants called in the genome between runs [8, 20, 29]. To determine whether a variant was synonymous or non-synonymous, CDS annotations of the Merlin strain were used in coordination with the variant caller. If a single nucleotide variant (SNV) occurred in the same position as a multi-nucleotide variant (MNV), the variants were reported separately, as the variants may be found in other case samples (CLC Genomics Workbench 10 (Qiagen)). The nomencla- ture is from http://www.hgvs.org/mutnomen/.
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Quantitative CMV PCR assay. The prototype AMPLICOR CMV MONITOR test, a quantitative microtiter-based PCR assay, was developed by Roche Mo- lecular Systems (Alameda, Calif.). CMV viral DNA in the specimen was quan- titated by coamplifying a region of the CMV DNA polymerase gene in the presence of a known quantity of quantitative standard. The primers used were specific for the CMV polymerase gene and amplified a 362-bp fragment of the gene (21). The test is designed with an internal quantitation standard (QS) that was constructed with the same primer sequence as the target DNA but with different intervening sequences. The unique probe sequence allows for the dif- ferentiation of QS DNA and target DNA. To ensure a similar amplification efficiency, the QS is the same size and has a similar G⫹C content as the CMV target region. The QS is added at a known concentration during specimen processing so that extraction and recovery of DNA, in addition to amplification and detection, can be monitored. The assay was performed according to the manufacturer’s recommendations. The lower limit of sensitivity of the assay is 400 copies/ml of plasma.
ABSTRACT The clinical utility of the QuantiFERON-CMV (QFN-CMV) assay in heart transplant recipients was assessed. Forty-four cytomegalovirus (CMV)-seropositive pa- tients were enrolled: 17 received antiviral prophylaxis, and 27 were managed preemptively. CMV-DNAemia monitoring was performed by the use of a quanti- tative real-time PCR assay. The QFN-CMV assay was retrospectively performed on blood samples collected at ﬁve posttransplant time points. A higher proportion of patients with an indeterminate QFN-CMV result after the suspension of prophylaxis than of patients who showed a global T-cell responsiveness developed CMV infec- tion (P ⫽ 0.036). Patients who reconstituted a CMV-speciﬁc response following the ﬁrst CMV-DNAemia-positive result (42.9%) showed a median CMV-DNAemia peak 1 log of magnitude lower than that seen with patients with indeterminate results, and all controlled viral replication spontaneously. The 25% of patients with an inde- terminate result developed CMV disease. In the preemptive strategy group, no dif- ferences in the development of subsequent infection, magnitude of viral load, and viral control were observed on the basis of QFN-CMV measurements performed be- fore and after the ﬁrst CMV-DNAemia-positive result. Considering both CMV preven- tion strategies, viral relapse was associated with the failure to reconstitute CMV- speciﬁc cell-mediated immunity (CMI) after the resolution of the ﬁrst episode of CMV infection (P ⫽ 0.032). QFN-CMV measurements can be a useful tool for identifying patients (i) at higher risk of developing infection after discontinuing antiviral prophy- laxis, (ii) with late CMV infection who would beneﬁt from appropriate antiviral inter- ventions, and (iii) at higher risk of viral relapses. QFN-CMV measurements taken within 1 month posttransplantation (early period) are not revealing.
CMV antigenemia has been previously shown to predict CMV disease in lung transplant recipients (10). In our patient population, which received intensive ganciclovir and CMVIG prophylaxis when CMV disease was anticipated, antigenemia testing had a sensitivity, specificity, PPV, and NPV of 48, 99, 85, and 98%, respectively. These results are in contrast with those reported by Egan et al. (10), who studied nine lung transplant patients, among others, and found that antigenemia testing had a 100% sensitivity and 93.7% specificity. The dif- ferences between the two studies with respect to the number of patients (41 versus 9), posttransplantation antiviral manage- ment (intense prophylaxis against CMV versus no prophylaxis at all), and duration of the observation period (12 months versus 8 months) might have contributed to these differences. In our study, CMV disease occurred in association with low and high numbers of positive cells, which implies that preemp- tive anti-CMV therapy needs to be entertained with any pos- itive antigenemia test result. On treatment, the number of positive cells rapidly decreased and the antigenemia test result became negative after 1 month of therapy for all patients, including those who later experienced a recurrence of the disease. Unlike PCR, antigenemia testing did not identify the patients at high risk of recurrent CMV.
It has been recently shown that cell-mediated immunity (CMI) is involved in augmented risk of congenital CMV trans- mission, particularly when high maternal CMI responses are associated with low maternal CMV IgG avidity (14). In this study, two interferon gamma (IFN- ␥ ) release assays (IGRA), the CMV enzyme-linked immunosorbent spot (ELISPOT) and CMV QuantiFERON assays, widely used to detect pathogen- specific CMI (15–21), were compared in a group of primarily and nonprimarily CMV-infected pregnant women and in a control group of healthy seropositive and seronegative preg- nant and nonpregnant women without evidence of active CMV infection. Several characteristics differ between the CMV ELISPOT and CMV QuantiFERON assays. The CMV ELISPOT assay is made on a given number of peripheral blood mononu- clear cells (PBMCs) (⬃2 ⫻ 10 5 ), while the CMV QuantiFERON
In our study, the quantitative results obtained with plasma (CMM) and PBLs (bDNA CMV 2.0) were highly significantly correlated ( ⫽ 0.92). A positive correlation has already been reported during acute visceral CMV disease (7, 29). Similarly, Zipeto et al. (37), Gerna et al. (14), and Boivin et al. (3, 4) have shown that the presence of CMV DNA in the plasma of HIV- positive patients usually reflected the presence of higher levels of CMV DNA in the corresponding PBLs. We observed few discordant results, and in most cases, when the results of two assays did not agree, the positive one was weakly positive while the other one was negative. Comparing a homemade PCR with PBLs and P-AMP with plasma, Boivin et al. (5) also found that discordant results were associated with low viral loads. In ad- dition, when we observed discordant results, the negative assay has often been positive previously and/or became positive later during the course of the CMV infection. Finally, when all the results were taken into consideration, the results of the assays were closely correlated.
Cytomegalovirus (CMV) is a major cause of disease in organ and cell transplant recipients (12, 25) and patients who are immunocompromised due to other clinical conditions, such as human immunodeficiency virus type 1 infection and chronic steroid use. Furthermore, CMV is associated with an increased risk of bacterial and fungal infections (11, 13, 31, 32) and graft rejection (8, 24), increased health care costs (9, 18), and de- creased survival (7, 10) in transplant recipients. While treat- ment of disease with specific anti-CMV drugs reduces the severity and mortality of CMV disease in transplant recipients (17, 29), prophylaxis and preemptive therapy are more effec- tive in preventing CMV disease (5, 6, 15, 21). The success of preemptive therapy in particular is dependent upon the avail- ability of sensitive, specific, and timely diagnostic tests for CMV infection. At our institutions, the CMV pp65 antigen- emia assay has been heavily relied upon for this purpose with considerable success (19, 20). However, the antigenemia assay is highly labor-intensive, not automated, susceptible to inaccu- racy when not performed on very fresh specimens, and subjec- tive in its reading.
Usher syndrome (USH) is a rare autosomal recessive group of disorders characterized by retinitis pigmentosa (RP), sensorineural hearing loss and vestibular dysfunc- tion. Three clinical types are distinguished (USH1, USH2 and USH3) and additional atypical subtypes have been described associated with 10 causative genes and three additional loci [1–5]. Hearing impairment is a first symp- tom to develop in USH. Visual symptoms manifest as ret- initis pigmentosa (RP), a progressive retinal dystrophy, with rod and secondary cone photoreceptor dysfunction and ultimate loss. USH is a clinically and genetically het- erogeneous disorder, making diagnosis and treatment challenging [6–9]. Previous studies categorized the
Two luciferase expressing viruses were constructed with the Towne CMV strain (Figure 1A). A recombinant b - galactosidase ( b -gal) CMV strain was first prepared as a backbone for luciferase CMV. Recombinant b -gal virus was isolated from positive cultures. This virus was used in a second-round DNA recombination to generate two luciferase-reporter CMV viruses: the luciferase gene being under the control of either UL54 (POL) or UL99 (pp28) promoters. Successful recombinants were isolated by loss of b-gal activity and the expression of luciferase protein. The loss of the b-gal gene and acquisition of the luciferase gene in the expected location was con- firmed by DNA sequencing (Genebank submission ID: 1420040, sequences are also available in Additional file 1). Insertion at the specific sites was verified by PCR sequencing (Figure 1B).
Acute retinal necrosis (ARN) is a rare, but potentially dev- astating, syndrome characterised by progressive peripheral necrotising retinitis. It was first described in 1971 , but it took more than a decade later for its herpetic aetiology to be discovered and antiviral therapy to become the mainstay of treatment [2,3]. The current standard of care for ARN consists of intravenous acyclovir 10 mg/kg (or 1500 mg/m 2 ) every 8 h for 5–10 days, followed by oral acyclovir 400-800 mg 5 times daily for an additional 6-12 weeks, to reduce the risk of second eye involvement . Nevertheless, there is a lack of consensus concerning the treatment of ARN, with an increasing number of groups reporting primary treatment of ARN with oral antiviral therapy alone [5-10], although others suggest that intravenous antiviral therapy is superior  or even that