Immune signatures underlying post-acute COVID-19 lung sequelae.

Severe coronavirus disease 2019 (COVID-19) pneumonia survivors often exhibit long-term pulmonary sequelae, but the underlying mechanisms or associated local and systemic immune correlates are not known. Here, we have performed high-dimensional characterization of the pathophysiological and immune traits of aged COVID-19 convalescents, and correlated the local and systemic immune profiles with pulmonary function and lung imaging. We found that chronic lung impairment was accompanied by persistent respiratory immune alterations. We showed that functional severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2)­specific memory T and B cells were enriched at the site of infection compared with those of blood. Detailed evaluation of the lung immune compartment revealed that dysregulated respiratory CD8+ T cell responses were associated with the impaired lung function after acute COVID-19. Single-cell transcriptomic analysis identified the potential pathogenic subsets of respiratory CD8+ T cells contributing to persistent tissue conditions after COVID-19. Our results have revealed pathophysiological and immune traits that may support the development of lung sequelae after SARS-CoV-2 pneumonia in older individuals, with implications for the treatment of chronic COVID-19 symptoms.

STAT Transcription Factors in T Cell Control of Health and Disease.

The Jak-STAT pathway is one of many pleiotropic signaling pathways that plays an important role in organismal development and in response to changing environmental cues. As a key signaling cascade for cytokines and growth factors, Jak-STAT plays central role in the innate and adaptive immune system. Cytokines control the stability, commitment, and maturation of cytotoxic and helper T cells, parts of the adaptive immune system that mediate immunity to pathogens and are linked to inflammatory diseases. Dysregulation of Jak-STAT protein expression or function leads to autoimmunity, allergic diseases, and cancer. Because of their central role in these responses, Jak and STAT molecules have been targeted to develop therapeutics. This review extensively discusses the mechanism of how Jak-STAT signaling in T cells defines our immune responses in the battle against foreign pathogens.

Helminth infection reactivates latent γ-herpesvirus via cytokine competition at a viral promoter.

Mammals are coinfected by multiple pathogens that interact through unknown mechanisms. We found that helminth infection, characterized by the induction of the cytokine interleukin-4 (IL-4) and the activation of the transcription factor Stat6, reactivated murine γ-herpesvirus infection in vivo. IL-4 promoted viral replication and blocked the antiviral effects of interferon-γ (IFNγ) by inducing Stat6 binding to the promoter for an important viral transcriptional transactivator. IL-4 also reactivated human Kaposi's sarcoma-associated herpesvirus from latency in cultured cells. Exogenous IL-4 plus blockade of IFNγ reactivated latent murine γ-herpesvirus infection in vivo, suggesting a "two-signal" model for viral reactivation. Thus, chronic herpesvirus infection, a component of the mammalian virome, is regulated by the counterpoised actions of multiple cytokines on viral promoters that have evolved to sense host immune status.

Induction of GITRL expression in human keratinocytes by Th2 cytokines and TNF-α: implications for atopic dermatitis.

BACKGROUND: Glucocorticoid-induced TNF receptor-related protein ligand (GITRL), a ligand for the T cell co-stimulatory molecule GITR, is expressed by keratinocytes and involved in chemokine production. The expression of GITRL in skin inflammation remains unknown. OBJECTIVES: This study investigated cytokine regulation of keratinocyte GITRL expression. METHODS: Glucocorticoid-induced TNF receptor expression was evaluated in cytokine-treated human epidermal keratinocytes (HEK)s, murine PAM 212 cell line, murine and human skin explants by real time PCR, flow cytometry and immunostaining. Functional responses to GITR fusion protein were examined by real time PCR and ELISA. GITRL expression in AD and psoriasis was studied by immunohistochemistry. RESULTS: Skin biopsies from STAT6VT transgenic mice, which develop spontaneous atopic skin inflammation, were found by immunofluoresence, to have increased keratinocyte GITRL expression. Exposure to Th2 cytokines augmented GITRL mRNA expression in the murine PAM 212 keratinocytic cell line and murine skin explants. In contrast, GITRL mRNA and protein expression was only increased in HEKs and human skin explants in the presence of the combination of TNF-α and Th2 cytokines. A synergistic effect of Th2 cytokines and GITR fusion protein on production of CCL17, the Th2 chemokine, by murine keratinocytes was demonstrated. Immunohistochemical staining showed that acute AD lesions have increased expression of GITRL compared with normal skin, chronic AD lesions and psoriatic plaques. CONCLUSIONS AND CLINICAL RELEVANCE: Our studies demonstrate that GITRL expression is augmented by Th2 cytokines and TNF-α in keratinocytes. Increased GITRL expression in acute AD skin lesions is shown. This observation suggests a link between cytokine-regulated keratinocyte GITRL expression and its role in inflammatory responses in AD.

Encoding a superantigen by Staphylococcus aureus does not affect clinical characteristics of infected atopic dermatitis lesions.

BACKGROUND: Bacterial infection with Staphylococcus aureus is a known trigger for the worsening of atopic dermatitis (AD). Staphylococcal superantigens have been theorized to make a potential contribution to this worsening of AD seen with infection. OBJECTIVES: We sought to assess whether encoding a superantigen by S. aureus affects the inflammatory characteristics of impetiginized AD skin lesions. METHODS: Fifty-two children with clinically impetiginized lesions of AD which were positive for S. aureus were enrolled in this study. A lesion was graded clinically using the Eczema Area and Severity Index (EASI), and then wash fluid was obtained from the lesion for quantitative bacterial culture, and measurement of bacterial products lipoteichoic acid and staphylococcal protein A and cytokines. The staphylococcal isolate was tested for antibiotic susceptibilities and the presence of a superantigen. RESULTS: Fifty-four per cent (28 of 52) of the staphylococcal isolates encoded a superantigen. The presence of a superantigen had no significant effect on EASI score, amounts of bacterial products or inflammatory cytokines in the AD lesion. CONCLUSIONS: These studies suggest that the expression of a superantigen by S. aureus alone does not play an important role in the increased skin inflammation associated with staphylococcal infection in childhood AD.

Chemokine receptor CCR5 Delta 32 genetic analysis using multiple specimen types and the NucliSens Basic Kit.

Resistance to HIV-1 infection and delayed disease progression have been associated with a 32-bp deletion (Delta32) in the gene encoding the CCR5 chemokine receptor. In the present study we describe the modification of a nucleic acid sequence-based amplification (NASBA)-based CCR5 genotyping assay for a NucliSens Basic Kit (Organon Teknika, Durham, N.C.) format using a new target-specific sandwich oligonucleotide detection methodology. The new method permitted the use of generic electrochemiluminescent probes supplied in the NucliSens Basic Kit, whereas the original NASBA method required expensive target-specific ruthenium detection probes. The Basic Kit CCR5 Delta32 genotypic analysis was in 100% concordance with both the original NASBA assay and DNA PCR results. This study also evaluated the use of multiple specimen types, including peripheral blood mononuclear cells (PBMC), whole blood, dried blood spots, buccal scrapings, and plasma, for CCR5 genotype analysis. The sensitivities of the three assays were comparable when PBMC or whole blood was the specimen source. In contrast, when dried blood spots, buccal scrapings, or plasma was used as the sample source, the sensitivity of DNA PCR was 80.95, 42.8, or 0%, respectively, compared to 100% sensitivity obtained with the original NASBA and Basic Kit NASBA assays. Our study indicates that the NucliSens Basic Kit NASBA assay is very sensitive and specific for CCR5 Delta32 genotyping using multiple sample types.

Role of interleukin-12 and stat-4 in the regulation of airway inflammation and hyperreactivity in respiratory syncytial virus infection.

Respiratory syncytial virus (RSV) is a respiratory pathogen that can cause significant morbidity in infants and young children. Interestingly, the majority of children who acquire a RSV infection do not exhibit severe symptoms. Development of a Th1 response has been associated with resolution of symptoms in viral infections and may explain mild RSV illness. The current study investigated the cytokine response observed in mild disease in C57BL/6 mice that had low airway resistance and mucus production with little pulmonary inflammation. RSV infection in these mice was accompanied by a fourfold increase in interleukin-12(IL-12). Treatment of RSV-infected mice with anti-IL-12 resulted in an increase in airway hyperreactivity, mucus production, and airway inflammation (eosinophilia). Since IL-12 activation is dependent on Stat-4-mediated intracellular signal transduction, similar experiments were performed in Stat-4 deficient mice and demonstrated similar results to those obtained from anti-IL-12 treated mice. Again, there was an increase in airway hyperreactivity and mucus production, and goblet cell hypertrophy. These studies support the importance of IL-12 in the immune response to RSV infection resulting in resolution of disease and protection from inappropriate inflammatory responses.

RANTES and MIP-1beta mRNA expression in human peripheral blood mononuclear cells: transcript quantification using NASBA technology.

The importance of chemokines in the immune response, as well as in a range of specific disease states, is becoming increasingly apparent. The role of CC- (or beta-) chemokines and their receptors in the pathology and mechanisms of HIV-1 infection has served to intensify interest in these factors. Although the functionality of these factors resides in their protein forms, assays for the detection and quantification of these protein factors in clinical samples are not readily available. Consequently, we designed NASBA-based assays for the quantification of the mRNA encoding two members of the CC-chemokine family: RANTES and MIP-1beta. The NASBA-based assays are extremely sensitive, accurate, and reproducible across a dynamic range of at least four orders of magnitude. Inter-assay performance is comparable to intra-assay performance. We applied these methods to the analysis of normal human PBMC and PBMC from HIV-1 infected individuals. Although MIP-1beta mRNA levels are higher than RANTES levels in both populations, RANTES levels in HIV-1+ patients are higher than in normal individuals. The utility of these assays in longitudinal studies of specific subpopulations of cells, as well as their potential use in clinical diagnostics, is discussed.

Cutting edge: differential requirements for Stat4 in expression of glycosyltransferases responsible for selectin ligand formation in Th1 cells.

A role for Stat4 in IL-12-induced up-regulation of selectin ligands on Th1 cells was explored. Th1 cells generated from Stat4(-/-) mice exhibited no IL-12-inducible P-selectin ligands, no up-regulation of core 2 beta1,6-glucosaminyltransferase I (C2GlcNAcT-I), and low levels of the Th1 transcription factor T-bet. In contrast, Stat4(-/-) Th1 cells exhibited only a partial defect in expression of IL-12-inducible E-selectin ligands and expressed equivalently high levels of alpha1,3-fucosyltransferase VII (FucT-VII) as wild-type Th1 cells. FucT-VII expression was induced by T cell activation, and was enhanced by IL-12 independently of Stat4, whereas C2GlcNAcT-I up-regulation was mediated exclusively by IL-12, acting through Stat4. These data show that FucT-VII and C2GlcNAcT-I are controlled through distinct pathways and imply the existence of at least one other IL-12-inducible glycosyltransferase required for E-selectin and possibly P-selectin ligand formation in Th1 cells.

Characterization of murine BATF: a negative regulator of activator protein-1 activity in the thymus.

BATF belongs to the AP-1/ATF superfamily of transcription factors and forms heterodimers with Jun proteins to bind AP-1 consensus DNA. Unlike Fos/Jun heterodimers which stimulate gene transcription, BATF/Jun heterodimers are transcriptionally inert and inhibit biological processes that are associated with the overstimulation of AP-1 activity. Here, we describe the murine BATF cDNA and genomic clones and map the BATF locus to chromosome 12 D2-3. Using in situ hybridization of BATF mRNA, we show that BATF gene expression is highly restricted, with the most prominent signals detected in the thymus. BATF mRNA levels are regulated differentially during discrete stages of T cell development and are up-regulated following activation of T cells in the periphery. To demonstrate the impact of BATF on AP-1 activity in vivo, AP-1 luciferase reporter mice were crossed to transgenic mice overexpressing BATF exclusively in thymic T cells. Results show that elevated levels of BATF protein correlate with reduced transactivation by AP-1. Since the differential regulation of AP-1 activity is linked to key transitions in the developing immune system, our observations support a critical role for BATF in determining the overall level of AP-1 activity, and thus AP-1 target gene expression, in specific T cell subtypes.

Transcriptional activation by a matrix associating region-binding protein. contextual requirements for the function of bright.

Bright (B cell regulator of IgH transcription) is a B cell-specific, matrix associating region-binding protein that transactivates gene expression from the IgH intronic enhancer (E mu). We show here that Bright has multiple contextual requirements to function as a transcriptional activator. Bright cannot transactivate via out of context, concatenated binding sites. Transactivation is maximal on integrated substrates. Two of the three previously identified binding sites in E mu are required for full Bright transactivation. The Bright DNA binding domain defined a new family, which includes SWI1, a component of the SWI.SNF complex shown to have high mobility group-like DNA binding characteristics. Similar to one group of high mobility group box proteins, Bright distorts E mu binding site-containing DNA on binding, supporting the concept that it mediates E mu remodeling. Transfection studies further implicate Bright in facilitating spatially separated promoter-enhancer interactions in both transient and stable assays. Finally, we show that overexpression of Bright leads to enhanced DNase I sensitivity of the endogenous E mu matrix associating regions. These data further suggest that Bright may contribute to increased gene expression by remodeling the immunoglobulin locus during B cell development.

Pivotal role of signal transducer and activator of transcription (Stat)4 and Stat6 in the innate immune response during sepsis.

Signal transducer and activator of transcription (Stat)4 and Stat6 are transcription factors that provide type 1 and type 2 response, respectively. Here, we explored the role of Stat4 and Stat6 in innate immunity during septic peritonitis. Stat4-/- and Stat6-/- mice were resistant to the lethality compared with wild-type (WT) mice. At the mechanistic level, bacterial levels in Stat6-/- mice were much lower than in WT mice, which was associated with increased peritoneal levels of interleukin (IL)-12, tumor necrosis factor (TNF)-alpha, macrophage-derived chemokine (MDC), and C10, known to enhance bacterial clearance. In Stat4-/- mice, hepatic inflammation and injury during sepsis were significantly ameliorated without affecting local responses. This event was associated with increased hepatic levels of IL-10 and IL-13, while decreasing those of macrophage inflammatory protein (MIP)-2 and KC. Sepsis-induced renal injury was also abrogated in Stat4-/- mice, which was accompanied by decreased renal levels of MIP-2 and KC without altering IL-10 and IL-13 levels. Thus, Stat6-/- and Stat4-/- mice appeared to be resistant to septic peritonitis by enhancing local bacterial clearance and modulating systemic organ damage, respectively, via balancing cytokine responses. These results clearly highlight an important role of local type 1 and systemic type 2 cytokine response in protective immunity during sepsis, which can be regulated by Stat proteins.

IL-13-induced airway hyperreactivity during respiratory syncytial virus infection is STAT6 dependent.

Airway damage and hyperreactivity induced during respiratory syncytial virus (RSV) infection can have a prolonged effect in infants and young children. These infections can alter the long-term function of the lung and may lead to severe asthma-like responses. In these studies, the role of IL-13 in inducing and maintaining a prolonged airway hyperreactivity response was examined using a mouse model of primary RSV infection. Using this model, there was evidence of significant airway epithelial cell damage and sloughing, along with mucus production. The airway hyperreactivity response was significantly increased by 8 days postinfection, peaked during days 10-12, and began to resolve by day 14. When the local production of Th1- and Th2-associated cytokines was examined, there was a significant increase, primarily in IL-13, as the viral response progressed. Treatment of RSV-infected mice with anti-IL-13 substantially inhibited airway hyperreactivity. Anti-IL-4 treatment had no effect on the RSV-induced responses. Interestingly, when IL-13 was neutralized, an early increase in IL-12 production was observed within the lungs, as was a significantly lower level of viral Ags, suggesting that IL-13 may be regulating an important antiviral pathway. The examination of RSV-induced airway hyperreactivity in STAT6(-/-) mice demonstrated a significant attenuation of the response, similar to the anti-IL-13 treatment. In addition, STAT6(-/-) mice had a significant alteration of mucus-producing cells in the airway. Altogether, these studies suggest that a primary factor leading to chronic RSV-induced airway dysfunction may be the inappropriate production of IL-13.

Stat4 regulates multiple components of IFN-gamma-inducing signaling pathways.

Stat4 is activated in response to IL-12. Most functions of IL-12, including the induction of IFN-gamma, are compromised in the absence of Stat4. Since the precise role of Stat4 in IFN-gamma induction has not been established, experiments were conducted to examine Stat4 activation of IFN-gamma and other genes required for cytokine-induced expression of IFN-gamma. We first examined IL-12 signaling components. Basal expression of IL-12Rss1 and IL-12Rss2 is decreased in Stat4-deficient cells compared with that in control cells. However, IL-12 was still capable of inducing equivalent phosphorylation of Jak2 and Tyk2 in wild-type and Stat4-deficient activated T cells. We have further determined that other cytokine signaling pathways that induce IFN-gamma production are defective in the absence of Stat4. IL-18 induces minimal IFN-gamma production from Stat4-deficient activated T cells compared with control cells. This is due to defective IL-18 signaling, which results from the lack of IL-12-induced, and Stat4-dependent, expression of the IL-18R. Following IL-12 pretreatment to induce IL-18R, wild-type, but not Stat4-deficient, activated T cells demonstrated IL-18-induced NF-kappaB DNA-binding activity. In addition, IL-12-pretreated Stat4-deficient activated T cells have minimal IFN-gamma production followed by stimulation with IL-18 alone or in combination with IL-12 compared with control cells. Thus, Stat4 activation by IL-12 is required for the function of multiple cytokine pathways that result in induction of IFN-gamma.

Cytokine-stimulated T lymphocyte proliferation is regulated by p27Kip1.

T lymphocyte growth is regulated by the cyclin-dependent kinase inhibitor p27(Kip1). Mice deficient in p27(Kip1) have increased proliferative responses to multiple cytokines, including IL-2, IL-4, and IL-12, but not to anti-CD3. In the absence of p27(Kip1), T cells proliferate faster than control cells, as evidenced by increased [(3)H]thymidine uptake, increased cell growth and division, and an increased number of cells in S phase. Importantly, this regulation is specific for p27(Kip1) in T cells, because hyperproliferation of T cells from mice deficient in p21(Cip1/Waf1) was not observed. In vivo, there is an expansion of activated/memory CD4(+) cells in p27(Kip1)-deficient mice before and after immunization. Furthermore, Ag-stimulated spleen cells from immunized p27(Kip1)-deficient mice demonstrated increased proliferative responses to IL-2 and increased secretion of IFN-gamma. Although IL-4 stimulated proliferative responses are diminished in Stat6-deficient T cells, activated T cells from mice doubly deficient in both p27(Kip1) and Stat6 recover normal proliferative responses to IL-4. Together, these data firmly support a role for p27(Kip1) as a negative regulator of cytokine-stimulated T cell growth.

Spontaneous production of C-C chemokines by individuals infected with human T lymphotropic virus type II (HTLV-II) alone and HTLV-II/HIV-1 coinfected individuals.

To investigate the immunological features of human T lymphotropic virus type II (HTLV-II) infection and specific mechanisms whereby HTLV-II might influence the progression of HIV-1 disease in coinfected individuals, we have analyzed the production of the C-C chemokines RANTES and macrophage inflammatory proteins 1alpha and 1alpha (MIP-1alpha and MIP-1beta) by PBMCs from HTLV-II-infected and HTLV-II/HIV-1-coinfected individuals. We observed spontaneous production of significant levels of MIP-1alpha and -1beta and, to a lesser extent, RANTES, from individuals infected with HTLV-II alone or with concomitant HIV-1 infection. Spontaneous C-C chemokine production was not observed in PBMCs from uninfected or HIV-1-infected individuals. Although HTLV-II is known to preferentially infect CD8+ lymphocytes in vivo, we observed that whereas RANTES was produced exclusively by the CD8+-enriched fraction, MIP-1alpha and -1beta were produced by both the CD8+-enriched and CD8+-depleted fractions of HTLV-II-infected PBMCs. RT-PCR demonstrated active expression of the HTLV-II regulatory protein Tax in the infected CD8+ T lymphocyte population, and it was further shown that Tax transactivates the promoters of MIP-1beta and RANTES. Therefore, it appears that HTLV-II stimulates the production of C-C chemokines both directly at a transcriptional level via the viral transactivator Tax and also indirectly. Although the HTLV-II-infected individuals in this study are all virtually asymptomatic, they certainly display an abnormal immune phenotype. Moreover, our findings suggest that HTLV-II, via chemokine production, would be expected to alter the progression of HIV-1 infection in coinfected individuals.

Imaging of Mycobacterium avium-intracellulare infection in AIDS patients on highly active antiretroviral therapy: reversal syndrome.

OBJECTIVE: We recently encountered six patients with AIDS and an unusual complication of disseminated infection with Mycobacterium avium-intracellulare, which developed after the initiation of highly active antiretroviral therapy, including protease inhibitors and two new nucleoside analogues. Each patient had a febrile illness after the initiation of therapy and then developed mass lesions containing mycobacterial organisms in various organ systems, including bone, skin, and mesenteric and mediastinal nodes. All these patients suddenly experienced improvement in immunologic status as evidenced by decreasing viral loads and increasing CD4 cell counts. We chose to call this reaction "M. avium-intracellulare reversal syndrome." We describe the radiologic appearance of this unusual manifestation of infection with M. avium-intracellulare in patients with AIDS. CONCLUSION: New or enlarging lymphadenopathy or unusual musculoskeletal and cutaneous infections in patients with AIDS who are receiving highly active antiretroviral therapy may represent a response of the recovering immune system to a new or previously subclinical infection with M. avium-intracellulare.

The p38 mitogen-activated protein kinase is required for IL-12-induced IFN-gamma expression.

IL-12 is a central immunoregulatory cytokine that promotes cell-mediated immune responses and the differentiation of naive CD4+ cells into Th1 cells. We and others have demonstrated that the Stat4 is critical for IFN-gamma production by activated T cells and Th1 cells. However, several studies have suggested that other pathways may be involved in IL-12-stimulated IFN-gamma expression. In this report we demonstrate that IL-12 activates mitogen-activated protein kinase kinase 3/6 (MKK) and p38 mitogen-activated protein kinase (MAPK), but not p44/42 (ERK) or stress-activated protein kinase/c-Jun N-terminal kinase MAPK. The activation of p38 MAPK is required for normal induction of IFN-gamma mRNA and IFN-gamma secretion by IL-12 in activated T cells and Th1 cells. Importantly, IL-12-stimulated p38 MAPK effector functions occur through a Stat4-independent mechanism and correlate with increased serine phosphorylation of activating transcription factor-2. The requirement for p38 MAPK in IL-12 function suggests that this pathway may be an important in vivo target for the anti-inflammatory actions of p38 MAPK inhibitors.

Cutting edge: differential expression of chemokines in Th1 and Th2 cells is dependent on Stat6 but not Stat4.

The in vivo function of Th cell subsets is largely dependent on the ability of differentiated CD4+ T cells to be recruited to specific sites and secrete restricted sets of cytokines. In this paper we demonstrate that Th1 and Th2 cells secrete discrete patterns of chemokines, small m.w. cytokines that function as chemoattractants in inflammatory reactions. Th2 cells secrete macrophage-derived chemokine and T cell activation gene 3, and acquisition of this pattern of expression is dependent on Stat6. In contrast, Th1 cells secrete lymphotactin and RANTES, though unlike IFN-gamma, expression of these chemokines is independent of Stat4. We further show that supernatants from activated Th2 cells preferentially induce the chemotaxis of Th2 over Th1 cells, corresponding with Stat6-dependent expression of CCR4 and CCR8 in Th2 cells. These data provide the basis for restricted and direct T cell-mediated cellular recruitment to sites of inflammation.

Detection of human cytomegalovirus pp67 late gene transcripts in cerebrospinal fluid of human immunodeficiency virus type 1-infected patients by nucleic acid sequence-based amplification.

This study examined the clinical correlation between the presence of human cytomegalovirus (HCMV) pp67 mRNA in cerebrospinal fluid (CSF) and active HCMV central nervous system (CNS) disease in patients with human immunodeficiency virus type 1 (HIV-1). In total, 76 CSF specimens collected from 65 HIV-1-positive patients diagnosed with HCMV CNS disease, other non-HCMV-related CNS diseases, or no CNS disease were tested for the presence of HCMV pp67 mRNA using the NucliSens cytomegalovirus (CMV) pp67 assay (Organon Teknika, Durham, N.C.). The results were compared to those of a nested PCR for the detection of HCMV glycoprotein B DNA and to those obtained by viral culture (54 samples). CSF specimens collected from patients without HCMV CNS disease yielded the following results: pp67 assay negative, 62 of 62 specimens; culture negative, 41 of 41 specimens; and PCR negative, 56 of 62 specimens (6 specimens were positive). CSF specimens collected from patients with HCMV CNS disease yielded the following results: pp67 assay positive, 9 of 13 specimens; PCR positive, 13 of 13 specimens; and culture positive, 2 of 13 specimens. After resolution of the discordant results, the following positive and negative predictive values (PPV and NPV, respectively) for the diagnosis of HCMV CNS disease were determined. The PPV for PCR, pp67 assay, and culture were 68.4, 100, and 100%, respectively, and the NPV for PCR, pp67 assay, and culture were 100, 97.0, and 82. 7%, respectively. The sensitivities for DNA PCR, pp67 assay, and culture for the detection of HCMV were 100, 84.6, and 18%, respectively, and the clinical specificities were 90.5, 100, and 100%, respectively. This study indicates that the detection of HCMV pp67 mRNA in CSF has good correlation with active HCMV CNS disease, whereas CSF culture is insensitive and qualitative DNA PCR may detect latent nonreplicating virus in CSF from patients without HCMV CNS disease.