Universal Newborn Screening for Congenital Cytomegalovirus Infection - From Infant to Maternal Infection: A Prospective Multicenter Study.

Introduction: Most infants at risk for cytomegalovirus (CMV)-associated sensorineural hearing loss (SNHL) are unrecognized because of the absence of a universal neonatal CMV screening. The search of CMV-DNA by molecular methods in salivary swabs was demonstrated to be a reliable approach. This study describes the results obtained by carrying out a universal screening for congenital CMV (cCMV) infection including all live-born newborns in three Italian sites, as well as the therapeutic interventions and clinical outcome of the CMV-infected neonates. Moreover, CMV maternal infection's characteristics were evaluated. Methods: To confirm or exclude cCMV infection, a CMV-DNA-positive result on a first salivary swab was followed by repeated saliva and urine samples collected within 21 days of age. Breast milk samples were also collected. The search of CMV-DNA was performed with a single automated quantitative commercial real-time PCR assay, regardless of the type of samples used. Results: A total of 3,151 newborns were enrolled; 21 (0.66%) of them were congenitally infected (median saliva viral load at screening, 6.65 [range, 5.03-7.17] log10 IU/ml). Very low/low viral load in screening saliva samples (median value, 1.87 [range, 1.14-2.59] log10 IU/ml) was associated with false-positive results (n = 54; 1.7%). CMV-DNA was detected in almost half of the breast milk samples of mother-infant pairs with a false-positive result, suggesting that contamination from breast milk may not be the only explanation in the study population. cCMV infection confirmation with the search of CMV-DNA in a urine sample proved to be the gold standard strategy, since false-positive results were observed in 4/54 (7.5%) of the repeated saliva samples. Symptomatic cCMV infection was observed in 3/21 (14.3%) infants; notably, one (4.7%) developed moderate unilateral SNHL at 5 months after birth. Finally, two symptomatic cCMV infections were associated with primary maternal infection acquired in the first trimester of gestation; one newborn with severe cCMV symptoms was born to a mother with no CMV checkups in pregnancy. Conclusion: Without universal neonatal CMV screening, some infected infants who develop late neurological sequelae may not be recognized and, consequently, they are not able to benefit early from instrumental and therapeutic interventions to limit and/or treat CMV disease.

Case Report: Molecular Detection of Dirofilaria repens in an Italian Patient after a Stay in Tanzania.

A 35-year-old man was admitted to a hospital in the south of Italy because of a periocular nodule and subpalpebral edema. The patient reported having been stayed in Tanzania five months before. Hematologic parameters were within the normality range, the Acanthocheilonema viteae ELISA did not detect significant levels of antifilarial IgG, and no further symptoms were described. The surgical inspection of the nodule led to the isolation of two filarioid parasites, identified as Dirofilaria repens by scanning electron microscope (SEM), and then by molecular assays. Knott's test did not reveal microfilaremia, whereas loop-mediated isothermal amplification and PCR detected D. repens DNA. The patient was treated with doxycycline, and he was found no more positive at the follow-up.

Chronic-Relapsing cutaneous leukocytoclastic vasculitis in a young patient with reduced EBV-specific T cell response using enzyme-linked immunospot (ELISPOT) assay successfully treated with Valaciclovir.

Among different pathogens, opportunistic viral infection caused by EBV is particularly relevant. This gammaherpesvirus, belonging to the Herpesviridae family, may complicate the disease course in different clinical settings by inducing pathological EBV pictures in patients with a defective immunologic response. Our report evaluated EBV-specific T cell responses by IFN- γ ELISPOT assay, which revealed defective EBV specific immunological response.

Zoster ... "a lmost" ... sine herpete: diagnostic utility of real time-polymerase chain reaction.

Zoster sine herpete is a particular form of varicella zoster virus (VZV) infection characterized by segmental pain and dysesthesia, without any cutaneous lesions ever becoming perceptible. This report describes the case of a female patient, presenting with intercostal pain associated with a single papulo-vesicular lesion localized within the same area. Thanks to such a lesion, real time-polymerase chain reaction (PCR) analysis on vesicle fluid swab was possible, thus revealing a significant number of VZV genome copies. This innovative tool has proven essential to diagnose this abortive form of herpes zoster, which would otherwise have remained unidentified.

Congenital toxoplasmosis and proposal of a new classification for the likelihood of primary maternal infection: analysis of 375 cases in Southeast Italy.

Objective: The aim of this study was to propose a classification in order to stratify the probability of an acute Toxoplasma infection in pregnancy and to estimate the risk of vertical transmission.Study design: We evaluated the likelihood of a primary maternal infection according to the Lebech classification and to the modified-Lebech classification proposed for our group of 375 patients referred for a suspected primary maternal infection. Fetal diagnosis included the examination of amniotic fluid by PCR to detect Toxoplasma DNA as a confirmation test.Results: Differences between the old and new classification resulted statistically significant for old classes defined as probable and unlikely with a clear shift of cases from the unlikely to the probable class in the new classification. Transmission rate showed a significant (p < .05) increase of the transmission rate in the probable class in our new classification as compared with the Lebech one.Conclusions: Results obtained in the present study suggest that the new IgG avidity-based classification herein proposed could estimate more precisely the likelihood of a primary maternal Toxoplasma infection as well as the risk of fetal infection, when compared with the historical Lebech Classification.

Leukocytoclastic Vasculitis Associated with HHV6-A/ciHHV6-A and HHV6-B Coinfection in an Immunocompetent Woman.

BACKGROUND AND OBJECTIVE: Leukocytoclastic vasculitis (LCV) is a small vessel vasculitis that can be limited to the skin but may also affect other organs. Often, its cause is unknown. LCV has previously been reported to occur with the reactivation of human herpesvirus 6 (HHV-6). Here, we report a second instance of HHV-6 reactivation in a 43-year-old woman with idiopathic cutaneous LCV. CASE DESCRIPTION: In this case, the patient was immunocompetent, and testing revealed that she had inherited chromosomally integrated human herpesvirus 6 variant A (iciHHV6-A) with a parallel skin infection of HHV-6B. The integrated ciHHV-6A strain was found to be transcriptionally active in the blood, while HHV-6B late antigen was detected in a skin biopsy. The patient's rash was not accompanied by fever nor systemic symptoms and resolved over four weeks without any therapeutic intervention. CONCLUSION: In light of the transcriptional activity documented in our case, further examination of a possible role for HHV-6 in the etiology of LCV is warranted.

Collaborative national multicenter for the identification of conversion factors from copies/mL to international units/mL for the normalization of HCMV DNA load.

The present multicentric (n = 11 laboratories) study aimed to identify conversion factors from copies/mL to international units (IU)/mL for the normalization of HCMV DNA load using the first WHO International Standard for HCMV nucleic acid amplification techniques and to enhance interlaboratory agreement of HCMV DNA quantification methods. Study protocols for whole blood and plasma (extraction and amplification) were performed to calculate conversion factors from HCMV DNA copy number to IU. The greatest variability was observed in samples with lower HCMV concentrations (3.0 Log10) in both biological matrices. Overall, 73.1% (206/282) of whole blood and 82.2% (324/394) of plasma samples analyzed fell within an acceptable variation range (±0.5 Log10 difference). An average of 0.64 (range 0.21-1.17) was the conversion factor calculated for the HCMV whole blood panel and 0.82 (range 0.39-2.2) for the HCMV plasma panel.

Serological survey on immunity status against polioviruses in children and adolescents living in a border region, Apulia (Southern Italy).

BACKGROUND: In 1988 the World Health Assembly adopted the goal to eradicate poliomyelitis by routine immunization using Oral Polio Vaccine (OPV). On 21 June 2002 the WHO European Region was declared polio-free. In 2008 poliomyelitis is still endemic in 4 countries (Nigeria, India, Pakistan, and Afghanistan), where 1201 new cases were registered in 2007; 107 sporadic cases were also notified in countries where poliovirus is not endemic. The aim of this work was to verify the level of antipoliomyelitis immunity status in children and adolescents in the Apulia region (south of Italy), which may be considered a border region due to its position. METHODS: 704 blood specimens from a convenience sample were collected in six laboratories. The age of subjects enrolled was 0-15 years. The immunity against poliomyelitis was evaluated by neutralizing antibody titration in tissue culture microplates. RESULTS: Seropositivity (neutralising antibodies titre > or = 8) for polioviruses 1, 2 and 3 was detected in 100%, 99.8% and 99.4% of collected sera. Antibody titres were not lower in subjects who received either four doses of inactivated polio vaccine (IPV) or a sequential schedule consisting of two doses of IPV and two of oral polio vaccine than in subjects who received four doses of OPV. CONCLUSION: These results confirmed current data of vaccine coverage for poliomyelitis: during the last ten years in Apulia, the coverage in 24 months old children was more than 90%. The high level of immunization found confirms the effectiveness both of the sequential schedule IPV-OPV and of the schedule all-IPV. Apulia region has to face daily arrivals of refugees and remains subject to the risk of the importation of poliovirus from endemic areas. Surveys aimed at determining anti-polio immunity in subpopulations as well as in the general population should be carried out.

Unsafe tap water in households supplied from groundwater in the Salento Region of Southern Italy.

Although the fractured aquifer of the Salento supplies over 80% of the drinking water requirements of the local population, its exposure to pollution has recently increased. In recent years, owing to the arid climate and droughts, the spreading of wastewater on soil for irrigation has become much more frequent. Consequently, hazardous and pathogenic microorganisms released with wastewater have been transported into the subsoil and have contaminated groundwater. An elaboration of epidemiological data has shown that the local population has the highest exposure to endemic gastroenteritis in Italy. In order to reduce human exposure to unsafe groundwater, the setback distance for drinking wells necessary to achieve the 'natural disinfection' criteria, has been determined experimentally at the Nardò aquifer (Salento region), supported by groundwater monitoring results and a mathematical transport model able to determine the apparent pathogenic microorganism pathways in fractures. The results also provided valuable inactivation constants of cultural indicators (coliforms, enterococci, Clostridium spores and somatic coliphages) and viruses in the wastewater that have been injected into the fractured aquifer since 1991. Furthermore, the efficacy of chlorine to remove viral indicators from water in a well 500 m from wastewater injection was tested. Hypochlorination reduces somatic coliphages and Clostridium spores in groundwaters but did not achieve complete inactivation in all tests. Complete disinfection of groundwater samples was possible only when there was an initial Clostridium spores count of < or = 10 CFU 100 ml (-1).

Atypical Herpetic Whitlow: A Diagnosis to Consider.

CONTEXT: Herpetic whitlow is caused by herpes virus (type1 or 2) during primary infection or as result of autoinoculation. Commonly, it is caused by HSV-2 in adults with positive history for genital infection. CASE DESCRIPTION: We report the case of a 44-year-old woman that came to our attention with a 3- year history of recurrent cutaneous eruption on the ring finger of her left hand associated to lymphangitis of the homolateral arm. Laboratory exams including PCR on blood and cutaneous swab allowed to diagnosis it as a rare case of herpetic whitlow. CONCLUSION: The case here reported demonstrates that herpetic whitlow should be kept in mind by physicians in recurrent cases of fingers infection. Advanced diagnostic techniques as PCR are required to help clinicians to achieve a definite diagnosis and to choose the right treatment.

Multicity Italian study of congenital cytomegalovirus infection.

BACKGROUND: Cytomegalovirus (CMV) infection is the most frequent congenital infection in humans. Its prevalence and the frequency of disabling sequelae must be assessed in different populations to permit the formulation or assessment of preventive measures. OBJECTIVES: To check the prevalence of congenital infection and seroprevalence in Italy; to verify the rate of sensorineural hearing loss (SNHL) in infected infants; and to assess the proportion of children with SNHL attributable to congenital CMV infection. METHODS: Diagnosis of congenital CMV infection was sought in 9032 children born between March 2002 and February 2003 by testing for viral DNA [CMV dried blood spot (DBS) test] in each newborn's Guthrie card and confirmation by isolation of CMV from urine collected in the first 3 weeks of life; CMV IgG testing in 1200 women of childbearing age; clinical and audiologic tests in the first 24 months for infected children; CMV DBS tests on the Guthrie cards collected from screening centers for 77 children (3 months-5 years) presenting SNHL of 40 dB or more. RESULTS: CMV infection was diagnosed in 14 asymptomatic and 2 symptomatic newborns (0.18%). CMV seroprevalence was 80%. In 2 infected infants, transient, unilateral SNHL was found. Nineteen of the 71 children with SNHL >70 dB were congenitally infected. CONCLUSIONS: The prevalence of congenital CMV infection is low in Italy. Population characteristics limiting the circulation of CMV strains in adult women might explain this. The fact that CMV contributes to significant SNHL highlights the need for preventive measures.

Epstein-barr virus induced cellular changes in nasal mucosa.

UNLABELLED: A 21-year-old man presented with nasal obstruction of the right nasal fossa of 1 year duration. Nasal endoscopy revealed in the right inferior turbinate head a rounded neoplasm about 1 cm in diameter. Cytologic study of a nasal scraping specimen disclosed numerous clusters containing columnar cells with cytomegaly, prominent multinucleation, markedly sparse shortened cilia; the cytoplasm contained an acidophil area and a small round area that stained poorly; cells with a large intracytoplasmic vacuole that was acidophil and PAS+. Serology tests using the nested polymer chain reaction (PCR) technique on serum, nasal and pharyngeal smears revealed an Epstein-Barr virus (EBV) infection that was confirmed at electron microscopy. The clinical and cytological features resolved 19 months after the initial evaluation. CONCLUSION: The authors advise carrying out clinical (endoscopy, serology, etc.) evaluation of all endonasal neoplasms and to routinely perform cytological study on nasal scraping specimens. When samples test positive for EBV, nasal and nasopharyngeal endoscopy should be performed regularly to detect possible evidence for nasopharyngeal carcinoma (NPC).

Nation-wide measure of variability in HCMV, EBV and BKV DNA quantification among centers involved in monitoring transplanted patients.

BACKGROUND: Inter-laboratory variability in quantifying pathogens involved in viral disease following transplantation may have a great impact on patient care, especially when pre-emptive strategies are used for prevention. OBJECTIVES: The aim of this study was to analyze the variability in quantifying CMV, EBV and BKV DNA from 15 virology laboratories of the Italian Infections in Transplant Working Group (GLaIT) involved in monitoring transplanted patients. STUDY DESIGN: Panels from international Quality Control programs for Molecular Diagnostics (QCMD, year 2012), specific for the detection of CMV in plasma, CMV in whole blood (WB), EBV and BKV were used. Intra- and inter-laboratory variability, as well as, deviations from QCMD consensus values were measured. RESULTS: 100% specificity was obtained with all panels. A sensitivity of 100% was achieved for EBV and BKV evaluations. Three CMV samples, with concentrations below 3 log10 copies/ml, were not detected by a few centers. Mean intra-laboratory variability (% CV) was 1.6 for CMV plasma and 3.0 for CMV WB. Mean inter-laboratory variability (% CV) was below 15% for all of the tested panels. Inter-laboratory variability was higher for CMV in WB with respect to the CMV plasma panel (3.0 vs 1.6% CV). The percentiles 87.7%, 58.6%, 89.6% and 74.7% fell within±0.5 log10 difference of the consensus values for CMV plasma, CMV WB, EBV and BKV panels, respectively. CONCLUSIONS: An acceptable intra- and inter-laboratory variability, in comparison with international standards was observed in this study. However, further harmonization in viral genome quantification is a reasonable goal for the future.

Recalcitrant scalp folliculitis: a possible role of herpes simplex virus type 2.

We report the case of a 70-year-old man with a 1 year history of relapsing folliculitis of the scalp. Bacteriological, mycological and the Tzanck tests from the lesions were negative. Histopathological study showed suppurative perifollicular flogosis. Virological cultures were negative, while HSV nested polymerase chain reaction (nPCR) assays made on swabs and histological sections from the scalp lesions demonstrated the presence of herpes simplex virus type-2 (HSV-2) in all samples. Skin swabs of healthy areas yielded negative results for HSV-2 infection. The folliculitis showed a marked and quick improvement after therapy with famciclovir suggesting a possible etiologic role of HSV-2 in the scalp folliculitis.

Modification of CMV DNA detection from dried blood spots for diagnosing congenital CMV infection.

BACKGROUND: Detection of viral DNA in dried blood spots using the Guthrie card (DBS test) is a reliable and practical method of diagnosing congenital cytomegalovirus (CMV) infection. The test lends itself to epidemiological studies to establish the prevalence of the infection, but also to neonatal screening for secondary prevention of sequelae. These applications would be facilitated if it were possible to use smaller samples and do the test on pools of individual cases. OBJECTIVE: To ascertain whether doing the test on smaller, pooled samples still accurately identifies neonates with congenital CMV infection. STUDY DESIGN: We tested DBS from: (A) 39 laboratory reference cases; (B) 156 neonates suspected of having congenital CMV infection; (C) 119 children examined for the retrospective diagnosis of congenital CMV; (D) mock specimens prepared with known amounts of viral DNA. RESULTS: The test using only one third of the usual amount of dried blood was 100% sensitive and specific compared to the standard DBS test (A) and to viral isolation (A and B). Pools of three single cases gave the same results as viral isolation (B) and the small-sample test (B and C). All the versions of the test gave a detection limit of 400 copies/ml. CONCLUSIONS: The modified procedure can accurately diagnose congenital CMV infection. It achieves savings in both the patient material and the costs of testing.

Relapsing herpes simplex-2 folliculitis in the beard area.

We describe the case of a 52-year-old immunocompetent man with recurrent folliculitis on the left cheek, associated with intense pain. Bacteriological, mycological and Tzanck tests from the lesions were negative. Histopathological study showed an aspecific flogosis pattern. Virological tests carried out on swabs and paraffin-embedded tissue sections from the facial lesions by nested PCR technique (nPCR) demonstrated the presence of herpes simplex virus type 2 (HSV\2) in both samples. Skin swabs from other healthy areas of the face resulted negative for herpetic infection. A diagnosis of recurrent herpetic folliculitis by HSV\2 was made. This case report underlines that even in immunocompetent patients HSV\2 lesions can feature atypical clinical aspects. In dermatological assessment the benefits of routine PCR techniques for differential diagnosis of herpetic infection should be considered above all for the prompt initiation of antiviral therapy and appropriate patient management.

High level immunity against poliomyelitis in African and Asian refugees in southern Italy.

The aim of this study was to evaluate the level of poliomyelitis immunization in refugees residing in the Asylum Seeker Center in Bari. The study was carried out during 2008 and involved 573 refugees. An antibody titer >or=1:8 was found in 99.6% for poliovirus 1, in 99.8% for poliovirus 2, and in 99.5% for poliovirus 3.