Clinical evaluation of TRCReady M.TB for rapid automated detection of M. tuberculosis complex in respiratory samples.

SETTING: Timely diagnosis of tuberculosis (TB) is essential for effectively controlling and managing the disease. Although international guidelines recommend acid-fast bacilli staining and culture as the 'gold standard', new molecular methods are available to safely and rapidly identify positive samples. OBJECTIVE: To evaluate the performance of the newer and fully automated version of a molecular assay for rRNA amplification (TRCReady® M.TB) on 1028 respiratory samples collected from 378 patients for its possible use as a reliable screening method. Results were evaluated using culture as the reference test. RESULTS: Of four diagnostic protocols employed, best results were obtained when TRCReady M.TB was used together with microscopy on the first respiratory sample, followed by microscopy alone on a second one. The sensitivity and specificity were respectively 97% and 100%, with a turnaround time of 24 h. We propose a possible laboratory algorithm for rapid identification of patients with TB. CONCLUSIONS: TRCReady offers the advantages of full automation and avoidance of cross-contamination. As such, it should be considered as a more economical option for TB screening than other commercial assays that are currently available.

The prevalence of antibodies to human herpesvirus 8 and hepatitis B virus in patients in two hospitals in Tanzania.

The aim of this study was to determine the seroprevalence of human herpesvirus 8 (HHV-8) and the immunization status for hepatitis B virus (HBV) infection in febrile patients in two districts of the United Republic of Tanzania. Between February and March 2007, blood samples were collected in Pemba Island and Tosamaganga from 336 outpatients and sent to the Virology Laboratory in Rome (Italy) for testing. HHV-8 DNA and HBV-DNA were amplified by two in-house molecular methods, anti-HHV-8 antibody titers were determined by an immunofluorescence assay (IFA), and anti-HCV, HBsAg, anti-HBs, and anti-HBc were evaluated by microplate enzyme immunoassay (MEIA). The seroprevalence of HHV-8 was 30.7% (96/313). In Pemba Island, the prevalence was lower than in Tosamaganga (14.4% vs. 46.3%). A higher prevalence of low titers of HHV-8 IgG (<1:80, 81%) was found among those under 5 years of age. HHV-8 DNA was detected in six seropositive patients (6.7%). The prevalence of HBsAg, anti-HBs, and anti-HBc was 4.3%, 37.6%, and 29.3%, respectively. Out of 277 patients, 70 had had a previous infection (25.3%). One case of occult hepatitis was found. The cover of hepatitis B vaccination was higher among children born after 2002 (66.7%) than in patients born before 2002. HHV-8 infection is endemic in Tanzania and the seroprevalence rate was higher in the mainland than on Pemba Island. The 3.9% percentage of HBsAg in children younger than 4 years of age suggests that increased efforts are required in order to achieve universal and compulsory immunization of children against HBV.

Cryptococcal lymphadenitis as a manifestation of immune reconstitution inflammatory syndrome in an HIV-positive patient: a case report and review of the literature.

Cryptococcus neoformans infections are typically associated with T-cell deficiencies, including acquired immunodeficiency syndrome (AIDS). Although highly active antiretroviral therapy (HAART) has strongly reduced AIDS-related opportunistic infections, the restoration and reactivation of CD4+ cells can induce an immune reconstitution inflammatory syndrome (IRIS), consisting in a deregulated inflammatory response to latent infectious pathogens and/or to their residual antigens. Cryptococcal lymphadenitis has occasionally been documented in IRIS. Here we report a case of histology- and culture-negative cryptococcal lymphadenitis associated with IRIS in an adult AIDS patient with a history of disseminated cryptococcosis, after the start of fully adherent HAART. Appropriate diagnosis was established on nested-PCR and sequence analysis of the interspacer region 2 of C. neoformans ribosomal DNA, and detection of slow-growing blastospores in enrichment cultures of fine-needle lymph node aspirate. Review of recent literature and our case findings suggest that IRIS-associated cryptococcal lymphadenitis is more likely the flare up of a latent infection rather than an immunopathological response to residual antigen of unviable cryptococci.

Imported malaria at Italy's National Institute for Infectious Diseases Lazzaro Spallanzani, 1984-2003.

Imported malaria is the most common cause of fatal infections in returning travellers. The increased amount of both tourist movement and migration has resulted in a growing number of people at risk of infection. In the present study, 507 malaria patients admitted to Italy's National Institute for Infectious Diseases in Rome between January 1984 and December 2003 were studied. Overall, 445 cases, or 87.7%, were acquired in Africa, of which 55% were acquired in five sub-Saharan countries. Plasmodium falciparum accounted for 393 (77.5%) of the imported cases. Patients consisted of short-term travellers (n = 213, 42%), long-term visitors (n = 134, 26.4%), and immigrants from endemic areas (n = 137, 27%). Malaria chemoprophylaxis was completed in less than one-quarter of all patients, with immigrants having the lowest rate of completion: only 3.6% of immigrants fully completed chemoprophylaxis compared to 31% of short-term travellers and 29.1% of long-term visitors (p < 0.001). Upon multivariate analysis, the lack of chemoprophylaxis was independently associated with the occurrence of severe malaria (p = 0.009). Severe malaria was reported in 59 (11.6%) individuals: all 11 deaths due to severe P. falciparum infection occurred in patients from sub-Saharan countries, two of whom were immigrants from countries where malaria is endemic. Malaria poses a serious health threat to individuals visiting endemic areas. Ensuring the correct chemoprophylaxis for all travellers, including immigrants from endemic areas, and providing prompt access to healthcare providers for unhealthy returning travellers are major points still to be addressed in Italy.

Atypical disseminated leishmaniasis resembling post-kala-azar dermal leishmaniasis in an HIV-infected patient.

Post-kala-azar dermal leishmaniasis (PKDL) is very uncommon among HIV-positive patients, and very few cases have so far been documented. A case of atypical disseminated leishmaniasis resembling PKDL in an HIV-positive patient successfully treated with N-methylglucamine antimoniate is reported. The polymerase chain reaction performed on the skin lesions was positive for Leishmania infantum.

Repetitive sequences upstream of the pfg27/25 gene determine polymorphism in laboratory and natural lines of Plasmodium falciparum.

The structure of the genomic region located upstream of the gametocyte-specific gene pfg27/25 of Plasmodium falciparum was analysed in laboratory lines and field isolates of the parasite. The gene is located in a subtelomeric region of chromosome 13 in parasite clones 3D7 and HB3. Analysis of laboratory lines and field isolates of P. falciparum indicated that polymorphism upstream of pfg27/25 is mainly due to the structure of a repetitive DNA region located at about half a kilobase from the pfg27/25 coding sequence. Different types of repetitive sequences are present in this region, whose copy number is variable in different parasite lines. In addition a GC-rich sequence element contained in this region, which is proposed to be the startpoint of pfg27/25 mRNA, presents either a direct or a reverse orientation in different parasite lines. Genomic deletions upstream of the pfg27/25 gene are also described in two laboratory lines of the parasite, which eliminate two newly identified malaria genes. orf P and orf Gap, from the genome of these parasites. One of them, orf Gap, deleted from the reference parasite clone 3D7, is abundantly expressed as mature mRNA in asexual parasites. PCR analysis on 64 field isolates of P. falciparum indicated that orf P and orf Gap sequences are present in all tested samples of naturally propagating parasites.

Genotyping of Plasmodium falciparum gametocytes by reverse transcriptase polymerase chain reaction.

A molecular assay has been developed for the specific detection and genetic characterisation of Plasmodium falciparum gametocytes in the blood of malaria infected individuals. The assay is based on the reverse transcription and polymerase chain reaction (RT-PCR) amplification of the messenger RNA of gene pfg377, a sexual-stage specific transcript abundantly produced in maturing gametocytes. The gene contains four regions of repetitive sequences, of which region 3 was shown to be the most polymorphic in laboratory clones and field isolates of the parasite. Analysis of samples of malaria infected blood by RT-PCR specific for region 3 has enabled identification of multiple gametocyte-producing clones within single infections. The assay is able to detect gametocytes below the threshold of microscopic detection, and is highly specific for its gametocyte targets also in the presence of a vast excess of asexual forms.

Cidofovir added to HAART improves virological and clinical outcome in AIDS-associated progressive multifocal leukoencephalopathy.

OBJECTIVES: To analyse the virological and clinical efficacy of cidofovir combined with highly active antiretroviral therapy (HAART) in AIDS-related progressive multifocal leukoencephalopathy (PML). DESIGN: Multicentre observational study of consecutive HIV-positive patients with histologically or virologically-proven PML. Group A, 26 patients treated with HAART; group B, 14 patients treated with HAART plus cidofovir 5 mg/kg intravenously per week for the first 2 weeks and alternate weeks thereafter. JC virus DNA was quantified in cerebrospinal fluid (CSF) by PCR. RESULTS: Baseline virological, immunological and clinical characteristics were homogeneous between the groups. In one case cidofovir was discontinued because of severe proteinuria. There was no significant difference in HIV RNA responses and changes in the number of CD4 cells between group A and B. After 2 months of therapy, five out of 12 (42%) patients from group A and seven out of eight (87%) from group B reached undetectable JC virus DNA in the CSF (Chi-square P = 0.04); moreover, 24% of group A and 57% of group B patients showed neurological improvement or stability (P = 0.038). One-year cumulative probability of survival was 0.67 with cidofovir and 0.31 without (log-rank test, P = 0.01). Variables independently associated with longer survival were the use of cidofovir, HAART prior to the onset of PML, a baseline JC virus DNA load in CSF < 4.7 log10 copies/ml, and a baseline Karnofsky performance status > or = 60. CONCLUSIONS: In AIDS-related PML, cidofovir added to HAART is associated with a more effective control of JCV replication, with improved neurological outcome and survival compared with HAART alone.

The effect of potent antiretroviral therapy and JC virus load in cerebrospinal fluid on clinical outcome of patients with AIDS-associated progressive multifocal leukoencephalopathy.

A multicenter analysis of 57 consecutive human immunodeficiency virus-positive patients with progressive multifocal leukoencephalopathy (PML) was performed, to identify correlates of longer survival. JC virus (JCV) DNA was quantified in the cerebrospinal fluid (CSF) by polymerase chain reaction. Two months after therapy, 4% of the patients without highly active antiretroviral therapy (HAART) and 26% with HAART showed neurologic improvement or stability (P=.03), and 8% and 57%, respectively, reached undetectable JCV DNA levels in the CSF (P=.04). One-year probability of survival was.04 without HAART and.46 with HAART. HAART and lack of neurologic progression 2 months after diagnosis were independently associated with longer survival. Among HAART-treated patients, a baseline JCV DNA <4.7 log, and reaching undetectable levels after therapy predicted longer survival. Survival of AIDS-related PML is improved by HAART when JCV replication is controlled.

gammadelta T-cell anergy in human immunodeficiency virus-infected persons with opportunistic infections and recovery after highly active antiretroviral therapy.

gammadelta T lymphocytes recognize non-peptidic microbial antigens without antigen processing and major histocompatibility complex (MHC) restriction, representing an early defence mechanism against invading pathogens. As a defective response to non-peptidic antigens was observed in human immunodeficiency virus-positive (HIV+) persons, the aims of this study were twofold: to analyse the incidence of gammadelta T-cell anergy in HIV+ positive patients with opportunistic infections/co-infections (HIV-OIC), and to investigate the role of highly active antiretroviral therapy (HAART) on gammadelta T-cell functions. Peripheral gammadelta T-cell distribution and in vitro reactivity to a non-peptidic mycobacterial antigen, isopentenyl pyrophosphate (IPP), were analysed. gammadelta T-cell subset distribution was altered more in HIV-OIC patients than in asymptomatic HIV+ subjects (HIV-ASY). Specifically, the Vdelta2/Vdelta1 ratio was inverted as a consequence of a decrease in Vdelta2 T-cell number. Moreover, IPP-stimulated Vdelta2 T cells from the HIV-OIC group displayed a major defect in interferon-gamma (IFN-gamma) production. Interestingly, HAART induced a sustained recovery of naive CD45RA+ and CD62L+ T cells and restored gammadelta T-cell function. Accordingly, in vitro CD45RA depletion resulted in gammadelta T-cell hyporesponsiveness. Altogether, the incidence of gammadelta T-cell anergy was increased in HIV-OIC patients and dependent on CD45RA helper function. Moreover, HAART was able to restore gammadelta T-cell reactivity, extending the immune recovery to non-peptide microbial antigens.

Hemoperfusion and liver disease.

After first adventurous attempts to apply perfusion techniques to the treatment of liver diseases, more extensive experiences have been acquired in the last twenty years, not only in acute hepatic failure but also in some chronic liver diseases (mainly in CAH:chronic active hepatitis, and in primary biliary cirrhosis) and in a severe complication of them, that is cryoglobulinemia. Some experiences on this field that are found in literature, using both plasma exchange and hemoperfusion, are reviewed and some personal data are reported: 15 patients with acute viral liver failure (survival rate: 33%) and 5 patients with a CAH-linked (3 viral and 2 autoimmune) cryoglobulinemia, in whom a good control of the disease parameters was obtained.

A case of falciparum malaria acquired in Italy.

The first case of Plasmodium falciparum malaria acquired in Italy after the eradication of the disease is reported. The P. falciparum strain was sensitive to chloroquine in vitro and in vivo. It seems most likely that an infective mosquito of tropical origin was responsible for transmission.