Paola Dal Monte

Clinical validation of Xpert MTB/RIF for the diagnosis of extrapulmonary tuberculosis

Extrapulmonary tuberculosis (EPTB) accounts for more than 20% of tuberculosis (TB) cases. Xpert MTB/RIF (Xpert) (Cepheid, Sunnyvale, CA, USA) is a fully automated amplification system, for which excellent results in the diagnosis of pulmonary TB in highly endemic countries have been recently reported. We aimed to assess the performance of the Xpert system in diagnosing EPTB in a low incidence setting. We investigated with Xpert a large number of consecutive extrapulmonary clinical specimens (1,476, corresponding to 1,068 patients) including both paediatric (494) and adult samples. We found, in comparison with a reference standard consisting of combination of culture and clinical diagnosis of TB, an overall sensitivity and specificity of 81.3% and 99.8% for Xpert, while the sensitivity of microscopy was 48%. For biopsies, urines, pus and cerebrospinal fluids the sensitivity exceeded 85%, while it was slightly under 80% for gastric aspirates. It was, in contrast, lower than 50% for cavitary fluids. High sensitivity and specificity (86.9% and 99.7%, respectively) were also obtained for paediatric specimens. Although the role of culture remains central in the microbiological diagnosis of EPTB, the sensitivity of Xpert in rapidly diagnosing the disease makes it a much better choice compared to smear microscopy. The ability to rule out the disease still remains suboptimal.

Intense Antiextracellular Adaptive Immune Response to Human Cytomegalovirus in Very Old Subjects with Impaired Health and Cognitive and Functional Status

Human aging is characterized by expanded and altered adaptive immune responses to human CMV (HCMV). It is unclear whether this expansion has its origins in age-related homeostatic disturbances or viral reactivation, whether anti-CMV immune surveillance may still be effective, and what are the consequences of this expanded immune response for health and longevity. We conducted an observational cross-sectional study in groups of HCMV-seropositive subjects aged ≥65 y of variable health status to compare the intensity of Ab responses against HCMV with those against EBV and with CD4+ and CD8+ T cell proinflammatory effector responses directed to HCMV-derived pp65 and immediate-early protein 1 synthetic peptides. Ab responses to HCMV, but not to EBV, and anti-HCMV CD4+, but not CD8+, T cell responses were more intense in elderly subjects aged ≥85 y in poor health and were inversely correlated with markers of functional activity and cognitive function. Therefore, humoral and CD4+ T cell anti-HCMV responses were specifically intensified in advanced aging associated with comorbidity and cognitive and functional impairments. Such a distinctive pattern of adaptive immunity indicates that immune responses targeting the extracellular phase of HCMV are increased in these elderly subjects and could represent an indirect effect of localized and undetectable HCMV reactivation. This study demonstrates that the oldest subjects in poor health with physical and mental impairment express intense functional immune responses to extracellular HCMV and suggests that they may be at risk for direct pathogenic effects by HCMV reactivation as well as indirect pathogenic effects linked to proinflammatory anti-HCMV effector responses.

Remodelling of the nuclear lamina during human cytomegalovirus infection: role of the viral proteins pUL50 and pUL53

A fundamental step in the efficient production of human cytomegalovirus (HCMV) progeny is viral egress from the nucleus to the cytoplasm of infected cells. In the family Herpesviridae, this process involves alteration of nuclear lamina components by two highly conserved proteins, whose homologues in HCMV are named pUL50 and pUL53. This study showed that HCMV infection induced the mislocalization of nuclear lamins and that pUL50 and pUL53 play a role in this event. At late stages of infection, both lamin A/C and lamin B showed an irregular distribution on the nuclear rim, coincident with areas of pUL53 accumulation. No variations in the total amount of nuclear lamins could be detected, supporting the view that HCMV induces a qualitative, rather than a quantitative, alteration of these cellular components, as has been suggested previously for other herpesviruses. Interestingly, pUL53, in the absence of other viral products, localized diffusely in the nucleus, whilst the co-expression and interaction of pUL53 with its partner, pUL50, restored its nuclear rim localization in distinct patches, thus indicating that pUL50 is sufficient to induce the localization of pUL53 observed during virus infection. Importantly, analysis of the nuclear lamina in the presence of pUL50-pUL53 complexes at the nuclear boundary and in the absence of other viral products showed that the two viral proteins were sufficient to promote alterations of lamins, strongly resembling those observed during HCMV infection. These results suggest that pUL50 and pUL53 may play an important role in the exit of virions from the nucleus by inducing structural modifications of the nuclear lamina.

Adenine glycosylase activity in mammalian tissues: an equivalent of ribosome‐inactivating proteins

Several plant tissues contain enzymes, provisionally called ribosome-inactivating proteins (RIPs), which damage ribosomes in an irreversible manner (reviewed in [1^3]) by removing a speci¢c adenine from the major rRNA. Subsequently it was observed that all RIPs remove adenine from DNA, and the denomination of polynucleotide:adenosine glycosidase was proposed for them [4], consistently with the o¤cial denomination of ricin (rRNA glycosidase, EC 3.2.2.22). In the present letter this denomination will be changed to adenine polynucleotide glycosylase (adenine glycosylase), by analogy with the denomination of DNA glycosylase used for similar enzymes. The presence of RIPs in diverse organisms led to the notion that equivalent proteins could exist in the animal kingdom [5]. We report now that partially puri¢ed preparations of some animal tissues remove adenine from DNA, and that their activity increases in stressed and virally infected cells. The crude extracts from several rat tissues did not exhibit any activity typical of RIPs; namely, they did not inhibit protein synthesis in a rabbit reticulocyte lysate nor caused any release of adenine from DNA. However, after the extracts were subjected to a chromatographic procedure on ProcionRed (Pharmacia) used to purify RIP, the extracts of rat spleens, lungs and brains exhibited a signi¢cant deadenylating activity (Table 1), still without any inhibitory activity on the reticulocyte lysate system at concentrations up to 100 Wg/ml of protein (results not shown). The rat spleens extracts were active also, in decreasing order, on poly(A), on rRNA (rat liver 28S+5S) and on tRNA. Much less or no activity at all was found in similarly treated extracts of mice, ox, hog, rabbit and human spleens. The activity was proportional to the concentration of extract present in the reaction mixture and had an optimum at pH 5.5^6.5 (optimum for plant RIPs acting on DNA is 4.0). When the semi-puri¢ed preparations were subjected to gel ¢ltration on a TSK-3000 column (Toso-Haas), the enzyme activity appeared in the fractions of the e¥uent corresponding to a molecular mass between 20 000 and 40 000. Also, the activity was bound to cation-exchange chromatography media in conditions of binding basic proteins (results not shown), suggestive of a protein with a high pI. Further puri¢cation of the enzyme activity from rat spleen extract was attempted on a micro-scale. After chromatography on Orange-matrix (Amicon), the activity was resolved in two peaks, although without a signi¢cant increase in speci¢c activity of either peak. Upon chromatography on mono-S ion exchange column (Pharmacia), the activity appeared widely distributed during a gradient elution, with a low yield, but with an increase of approx. 10-fold of speci¢c activity in selected fractions. These results indicate that these enzymes may be present in di¡erent isoforms, as it happens with plant RIPs. Fractions with higher speci¢c activity were subjected to analysis of reverse-phase chromatography, still showing a substantial heterogeneity of protein species. Rat spleen extracts enriched by Procion-Red chromatography had an activity on calf thymus DNA lower than on herring sperm-DNA, and had no activity at all on deoxy-adenosine or 3PdAMP and 5PdAMP (results not shown).

First independent evaluation of QuantiFERON-TB Plus performance

Tuberculosis elimination requires an effective strategy to diagnose and treat people infected with Mycobacterium tuberculosis who would otherwise be at high risk of developing and transmitting active disease [1, 2]. The diagnostic tools for latent tuberculosis infection (LTBI) are the tuberculin skin test (TST) and the T-cell interferon-γ release assays (IGRAs). Two IGRAs are commercially available, QuantiFERON-TB Gold In-Tube (QFT-GIT) (Qiagen, Hilden, Germany) and T-SPOT.TB (Oxford Immunotec, Abingdon, UK). Compared to the TST, IGRAs offer operational advantages and higher specificity in the bacille Calmette–Guérin (BCG)-vaccinated population [3], and they are at least as sensitive for LTBI [4]. However, IGRAs have limitations: reduced sensitivity in children and immunocompromised subjects, including HIV-infected individuals [3, 4]; failure to discriminate between active tuberculosis and LTBI; and poor correlation with the risk of progression to active disease [3]. QuantiFERON-TB Plus improves sensitivity for active TB and maintains high specificity among unvaccinated controls http://ow.ly/XjYPK

Intrauterine cytomegalovirus infection and glycoprotein N (gN) genotypes.

BACKGROUND Human cytomegalovirus (HCMV) clinical isolates display genetic polymorphisms, supposed to be related with strain-specific tissue-tropism and HCMV-induced immunopathogenesis. One recently discovered polymorphic gene is ORF UL73, encoding for the envelope glycoprotein gN. Among HCMV clinical strains, it shows four distinct genomic variants denoted as gN-1, gN-2, gN-3 and gN-4. OBJECTIVES Aims of this study were to assess the prevalence of the different gN types in the populations examined and to investigate the possible relationship between genotypes and severity of congenital CMV disease. STUDY DESIGN The gN genotyping was carried out by sequencing analysis of the HCMV ORF UL73. Comparisons were made by chi-square test and contingency tables. RESULTS All the four gN genotypes can cause congenital infections and the overall distribution was as follows: gN-1, 23.6%; gN-2, 1.1%; gN-3, 12.9%; gN-4, 62.4%. None of them seems to be preferentially associated with vertical transmission or with acute outcome of congenital infection. However, considering the chronic outcome and long-term sequelae, there was a statistically significant (P<0.05) difference between congenitally infected infants with or without adverse chronic outcome. CONCLUSIONS HCMV congenital infections, which displayed a prevalence of the gN-1 variants, seem to be associated with favorable chronic outcome.

Performance of interferon-γ release assay for the diagnosis of active or latent tuberculosis in children in the first 2 years of age: a multicenter study of the Italian Society of Pediatric Infectious Diseases.

Background: The diagnosis of latent or active tuberculosis in children is often challenging. Recently, interferon-&ggr; release assays have been licensed, but their diagnostic accuracy in young children remains questionable as frequent false-negative or indeterminate results have been reported. Methods: We performed a multicenter, retrospective study in children 0–24 months of age who were tested at least once with QuantiFERON-TB Gold-in-tube (QTF-IT) ± tuberculin skin test (TST), to analyze its use and performance in clinical practice. Results: Eight-hundred and twenty-three children (449 males, median age 13.5 months) were enrolled. QTF-IT sensitivity and specificity for active tuberculosis were 92.4% and 98.6%, respectively. Indeterminate tests (4.2 %) were not related to age (P = 0.838) or gender (P = 0.223); 32 children (91.4 %) with an indeterminate QTF-IT ultimately resulted uninfected. In the 616 subjects with valid paired results of QTF-IT and TST, sensitivity and specificity were comparable (91.1% vs. 85.1% and 98.1% vs. 97.9%, respectively). Diagnostic concordance between tests was higher in Bacillus Calmétte-Guerin nonvaccinated children (&kgr; = 0.802). A high rate of discordant tests was observed in latent infections. Conclusions: QTF-IT showed good sensitivity and specificity, and a low rate of indeterminate results in the first 2 years of life, supporting its use at this age. However, considering costs and the similar performance between QTF-IT and TST, it is reasonable to suggest the latter as first-line testing in young children. The complementary use of TST and interferon-&ggr; release assays may be considered in selected cases to improve the accuracy of testing.

Human Cytomegalovirus Carries a Cell-Derived Phospholipase A2 Required for Infectivity

ABSTRACT Human cytomegalovirus (HCMV) is known to carry host cell-derived proteins and mRNAs whose role in cell infection is not understood. We have identified a phospholipase A2 (PLA2) activity borne by HCMV by using an assay based on the hydrolysis of fluorescent phosphatidylcholine. This activity was found in all virus strains analyzed and in purified strains. It was calcium dependent and was sensitive to inhibitors of cytosolic PLA2 (cPLA2) but not to inhibitors of soluble PLA2 or calcium-independent PLA2. No other phospholipase activity was detected in the virus. Purified virus was found to contain human cellular cPLA2α, as detected by monoclonal antibody. No homology with PLA2 was found in the genome of HCMV, indicating that HCMV does not code for a PLA2. Decreased de novo expression of immediate-early proteins 1 and 2 (IE1 and IE2), tegument phosphoprotein pp65, and virus production was observed when HCMV was treated with inhibitors of cPLA2. Cell entry of HCMV was not altered by those inhibitors, suggesting the action of cPLA2 was postentry. Together, our results indicate a selective sorting of a cell-derived cPLA2 during HCMV maturation, which is further required for infectivity.

Diagnosis of smear-negative tuberculosis is greatly improved by Xpert MTB/RIF

Background Diagnosis of pulmonary (PTB) and extra-pulmonary tuberculosis (EPTB) in smear-negative patients can be difficult. We assessed retrospectively the performance of Xpert MTB/RIF system (Xpert, Cepheid) in diagnosing smear-negative tuberculosis (TB), which represents the most common form of TB in a low incidence setting. Methods Performance of Xpert was compared to acid-fast microscopic examination using Ziehl-Neelsen (ZN) stain in patients with culture-confirmed TB. Results 386 Mycobacterium tuberculosis (MTB) culture-positive samples were detected out of 5170 specimens tested with smear microscopy, Xpert and culture: 323 were both culture- and Xpert-positive, and 63 culture-positive only. Of these, 234 (60.6%) were smear-negative. In addition Xpert detected 40 probable TB cases, based on clinical findings, which were culture-negative. Compared to culture, Xpert showed an overall sensitivity of 83.7% and a specificity of 99.1%; sensitivity was higher for respiratory samples (86.5%) than for non-respiratory samples (76.8%). Xpert sensitivity for smear-negative culture-confirmed TB was 73.1% and was not influenced by TB localization. As sensitivity of microscopy alone was poor (39.4%), Xpert improved both diagnosis of pulmonary TB (Δ = 36.5%) and extra-pulmonary TB (Δ = 63.4%). Conclusions Xpert MTB/RIF is a sensitive method for rapid diagnosis of TB compared to the conventional ZN staining. Xpert can serve as a sensitive and time-saving diagnostic method for microbiological diagnosis of smear-negative TB in countries with a low TB prevalence.

Latency‐associated human cytomegalovirus glycoprotein N genotypes in monocytes from healthy blood donors

BACKGROUND: The β‐herpesvirus human cytomegalovirus (HCMV) infects a variety of cell types and maintains a lifelong relationship with its host by way of a latent infection in circulating monocytes, myeloid precursor cells, and the hematopoietic progenitor population. Viral strain heterogeneity, shown by gene polymorphisms, has been implicated in the majority of HCMV biologic behaviors. HCMV UL73 encodes the polymorphic envelope glycoprotein N (gN), which shows seven genotypes (gN‐1, gN‐2, gN‐3a, gN‐3b, gN‐4a, gN‐4b, and gN‐4c).