Sara Calattini

Clinical Use of Polymerase Chain Reaction Performed on Peripheral Blood and Bone Marrow Samples for the Diagnosis and Monitoring of Visceral Leishmaniasis in HIV-Infected and HIV-Uninfected Patients : A Single-Center, 8-Year Experience in Italy and Review of the Literature

BACKGROUND To overcome some of the limitations of conventional microbiologic techniques, polymerase chain reaction (PCR)-based assays are proposed as useful tools for the diagnosis of visceral leishmaniasis. PATIENTS AND METHODS A comparative study using conventional microbiologic techniques (i.e., serologic testing, microscopic examination, and culture) and a Leishmania species-specific PCR assay, using peripheral blood and bone marrow aspirate samples as templates, was conducted during an 8-year period. The study cohort consisted of 594 Italian immunocompetent (adult and pediatric) and immunocompromised (adult) patients experiencing febrile syndromes associated with hematologic alterations and/or hepatosplenomegaly. Identification of the infecting protozoa at the species level was directly obtained by PCR of peripheral blood samples, followed by restriction fragment-length polymorphism analysis of the amplified products, and the results were compared with those of isoenzyme typing of Leishmania species strains from patients, which were isolated in vitro. RESULTS Sixty-eight patients (11.4%) had a confirmed diagnosis of visceral leishmaniasis. Eleven cases were observed in human immunodeficiency virus (HIV)-uninfected adults, 20 cases were observed in HIV-infected adults, and the remaining 37 cases were diagnosed in HIV-uninfected children. In the diagnosis of primary visceral leishmaniasis, the sensitivities of the Leishmania species-specific PCR were 95.7% for bone marrow aspirate samples and 98.5% for peripheral blood samples versus sensitivities of 76.2%, 85.5%, and 90.2% for bone marrow aspirate isolation, serologic testing, and microscopic examination of bone marrow biopsy specimens, respectively. None of 229 healthy blood donors or 25 patients with imported malaria who were used as negative control subjects had PCR results positive for Leishmania species in peripheral blood samples (i.e., specificity of Leishmania species-specific PCR, 100%). PCR and restriction fragment-length polymorphism analysis for Leishmania species identification revealed 100% concordance with isoenzyme typing in the 19 patients for whom the latter data were available. CONCLUSIONS PCR assay is a highly sensitive and specific tool for the diagnosis of visceral leishmaniasis in both immunocompetent and immunocompromised patients and can be reliably used for rapid parasite identification at the species level.

Role of PCR in Diagnosis and Prognosis of Visceral Leishmaniasis in Patients Coinfected with Human Immunodeficiency Virus Type 1

ABSTRACT A group of 76 consecutive human immunodeficiency virus (HIV)-positive patients with fever of unknown origin (n = 52) or fever associated with pulmonary diseases was evaluated in order to assess the usefulness of PCR with peripheral blood in the diagnosis and follow-up of visceral leishmaniasis. We identified 10 cases of visceral leishmaniasis among the 52 patients with fever of unknown origin. At the time of diagnosis, all were parasitemic by PCR with peripheral blood. During follow-up, a progressive decline in parasitemia was observed under therapy, and all patients became PCR negative after a median of 5 weeks (range, 6 to 21 weeks). However, in eight of nine patients monitored for a median period of 88 weeks (range, 33 to 110 weeks), visceral leishmaniasis relapsed, with positive results by PCR with peripheral blood reappearing 1 to 2 weeks before the clinical onset of disease. Eight Leishmania infantum and two Leishmania donovani infections were identified by PCR-restriction fragment length polymorphism analysis. PCR with peripheral blood is a reliable method for diagnosis of visceral leishmaniasis in HIV-infected patients. During follow-up, it substantially reduces the need for traditional invasive tests to assess parasitological response, while a positive PCR result is predictive of clinical relapse.

Simian Foamy Virus Transmission from Apes to Humans, Rural Cameroon

Bites from apes efficiently transmit the foamy virus to humans in natural settings in central Africa.

Human T-Cell Lymphotropic Virus Type 3: Complete Nucleotide Sequence and Characterization of the Human Tax3 Protein

ABSTRACT We and others have recently uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3), the third member of the HTLV family. We have now sequenced the full-length HTLV-3Pyl43 provirus. As expected, HTLV-3Pyl43 contains open reading frames corresponding to the gag, pol, env, tax, and rex genes. Interestingly, its long terminal repeat (LTR) includes only two Tax-responsive elements, as is the case for type 3 simian T-cell lymphotropic viruses (STLV-3). Phylogenetic analyses reveal that HTLV-3Pyl43 is closely related to central African STLV-3. Unexpectedly, the proximal pX region of HTLV-3Pyl43 lacks 366 bp compared to its STLV-3 counterpart. Because of this deletion, the previously described RorfII sequence is lacking. At the amino acid level, Tax3Pyl43 displays strong similarities with HTLV-1 Tax, including the sequence of a PDZ class I binding motif. In transient-transfection assays, Tax3Pyl43 activates the transcriptions from HTLV-3, HTLV-1, and HTLV-2 LTRs. Mutational analysis indicates that two functional domains (M22 and M47) important for transactivation through the CREB/ATF or NF-κB pathway are similar but not identical in Tax1 and Tax3Pyl43. We also show that Tax3Pyl43 transactivates the human interleukin-8 and Bcl-XL promoters through the induction of the NF-κB pathway. On the other hand, Tax3Pyl43 represses the transcriptional activity of the p53 tumor suppressor protein as well as the c-Myb promoter. Altogether, these results demonstrate that although HTLV-3 and HTLV-1 have only 60% identity, Tax3Pyl43 is functionally closely related to the transforming protein Tax1 and suggest that HTLV-3, like HTLV-1, might be pathogenic in vivo.

Prospective observational study of fever in hospitalized returning travelers and migrants from tropical areas, 1997-2001.

BACKGROUND An estimated 50 million people each year from industrialized countries visit tropical areas: 3% to 11% of these travelers report a febrile illness on their return. We conducted a 5-year prospective observational study on the causes of fever in patients admitted to a university teaching hospital after returning from the tropics. METHODS We enrolled in this study all consecutive patients admitted to the Division of Infectious Diseases of the University of Milan, Italy, between January 1997 and December 2001 presenting with fever (oral temperature > or =37.5 degrees C) and a history of travel to a tropical country in the previous 6 months. RESULTS Seven percent (147/2,074) of all hospital admissions in the study period were due to fever in travelers and migrants returning from the tropics. Malaria accounted for 47.6 % of all admissions (70/147), followed by presumed self-limiting viral infections (12%). Pretravel screening and vaccination strategies could have prevented a considerable number of hospitalizations (e.g., hepatitis A and typhoid fever). The most useful investigations were blood examination and PCR for malaria, which gave positive results in 65% of cases in which they were performed. CONCLUSIONS During a 5-year period, the number of patients returning from tropical areas who were admitted with fever to a university hospital in northern Italy remained stable; malaria was the most frequent diagnosis, and should be considered in any febrile patient returning from the tropics. With the exception of hepatitis A and dengue fever infections, in a real-world setting serology is of modest utility and is probably overused.

Seroprevalence and Molecular Epidemiology of Human T-Cell Leukemia Virus Type 1 (HTLV-1) and HTLV-2 in Blood Donors from Dakar, Senegal

ABSTRACT In 2002, human T-cell leukemia virus type 1 (HTLV-1) and HTLV-2 seroprevalence was 0.16% (8/4,900) in blood donors from Dakar, Senegal. Most of the positive donors originated from the country’s southern region. Seven donors were infected by HTLV-1 (of cosmopolitan subtype), and one was infected by HTLV-2. These data highlight the problem of transfusion safety in this area where HTLV-1-associated lymphoproliferative and neurological diseases are endemic.

HTLV-2B Strains, Similar to Those Found in Several Amerindian Tribes, Are Endemic in Central African Bakola Pygmies

BACKGROUND The presence and origin of endemic foci of human T-lymphotropic virus type 2 (HTLV2) infection in Africa remain a matter of debate. METHODS To better appreciate such determinants, we performed a survey of 1918 inhabitants from Cameroon forest areas, including 1051 Bakola Pygmies and 867 Bantus. RESULTS The overall HTLV-1/2 seroprevalence was 4% (49 cases of HTLV-1 and 27 cases of HTLV-2 infection). Both infections were mainly restricted to the Bakola Pygmies, with surprisingly no HTLV-2 infections in the Bantu population. Both HTLV-1 and HTLV-2 seroprevalences increased with age. There was evidence of ongoing HTLV-2 transmission in this population. Lymphoid T cell lines producing HTLV-2 were established. HTLV-2 long terminal repeat sequences (672 base pairs) obtained from 7 infected Bakola were highly similar to each other (<1% nucleotide divergence), as well as to Amerindian HTLV-2B strains. Analyses on a complete sequence (8954 base pairs) confirmed that it was a typical HTLV-2 subtype B strain. Along with molecular clock analysis, these data strongly suggest that HTLV-2 has been endemic in the Bakola Pygmy population for a long time. CONCLUSIONS This study demonstrates clearly an HTLV-2 endemicity with ongoing transmission in an African population. Furthermore, it give insights into central questions regarding the origins and evolution rate of HTLV-2 and the migrations of infected populations.

Emergence of simian foamy viruses in humans: facts and unanswered questions

A large proportion of viral pathogens that have emerged in humans are considered to have originated in animals. Simian viral infections of humans represent an increasing public health concern. This is well illustrated by retroviruses such as HIV-1/2 and human T-cell lymphotropic virus (HTLV)-1, which have a unique ability to cross species, adapt to a new host and spread. In this short review, we will present the currently available data on the transmission of the simian foamy retroviruses (SFVs) to humans. Indeed, recent data indicate the presence of these exogenous retroviruses, of the Spumaretrovirinae subfamily and of the Spumavirus genus, in individuals occupationally exposed to nonhuman primates (animal caretaker, veterinarian, zoo worker) and in individuals having contact with apes and monkeys, such as hunters in Central Africa. The main unanswered questions concerning the natural history of such SFVs in humans, for instance, their magnitude and geographical distribution, their interhuman transmissi...

Construction and Characterization of a Full-Length Infectious Simian T-Cell Lymphotropic Virus Type 3 Molecular Clone

ABSTRACT Together with their simian T-cell lymphotropic virus (STLV) equivalent, human T-cell lymphotropic virus type 1 (HTLV-1), HTLV-2, and HTLV-3 form the primate T-cell lymphotropic virus (PTLV) group. Over the years, understanding the biology and pathogenesis of HTLV-1 and HTLV-2 has been widely improved by the creation of molecular clones. In contrast, so far, PTLV-3 experimental studies have been restricted to the overexpression of the tax gene using reporter assays. We have therefore decided to construct an STLV-3 molecular clone. We generated a full-length STLV-3 proviral clone (8,891 bp) by PCR amplification of overlapping fragments. This STLV-3 molecular clone was then transfected into 293T cells. Reverse transcriptase PCR experiments followed by sequence analysis of the amplified products allowed us to establish that both gag and tax/rex mRNAs were transcribed. Western blotting further demonstrated the presence of the STLV-3 p24gag protein in the cell culture supernatant from transfected cells. Transient transfection of 293T cells and of 293T-long terminal repeat-green fluorescent protein cells with the STLV-3 clone promoted syncytium formation, a hallmark of PTLV Env expression, as well as the appearance of fluorescent cells, also demonstrating that the Tax3 protein was expressed. Virus particles were visible by electron microscopy. These particles are infectious, as demonstrated by our cell-free-infection experiments with purified virions. All together, our data demonstrate that the STLV-3 molecular clone is functional and infectious. This clone will give us a unique opportunity to study in vitro the different pX transcripts and the putative presence of antisense transcripts and to evaluate the PTLV-3 pathogenicity in vivo.

Construction and Characterization of a Human T-Cell Lymphotropic Virus Type 3 Infectious Molecular Clone

ABSTRACT We and others have uncovered the existence of human T-cell lymphotropic virus type 3 (HTLV-3). We have now generated an HTLV-3 proviral clone. We established that gag, env, pol, pro, and tax/rex as well as minus-strand mRNAs are present in cells transfected with the HTLV-3 clone. HTLV-3 p24gag protein is detected in the cell culture supernatant. Transfection of 293T-long terminal repeat (LTR)-green fluorescent protein (GFP) cells with the HTLV-3 clone promotes formation of syncytia, a hallmark of Env expression, together with the appearance of fluorescent cells, demonstrating that Tax is expressed. Viral particles are visible by electron microscopy. These particles are infectious, as demonstrated by infection experiments with purified virions.