Giovanna Bestetti

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.

A third genotype of the human parvovirus PARV4 in sub-Saharan Africa.

PARV4 is a recently discovered human parvovirus widely distributed in injecting drug users in the USA and Europe, particularly in those co-infected with human immunodeficiency virus (HIV). Like parvovirus B19, PARV4 persists in previously exposed individuals. In bone marrow and lymphoid tissue, PARV4 sequences were detected in two sub-Saharan African study subjects with AIDS but without a reported history of parenteral exposure and who were uninfected with hepatitis C virus. PARV4 variants infecting these subjects were phylogenetically distinct from genotypes 1 and 2 (formerly PARV5) that were reported previously. Analysis of near-complete genome sequences demonstrated that they should be classified as a third (equidistant) PARV4 genotype. The availability of a further near-complete genome sequence of this novel genotype facilitated identification of conserved novel open reading frames embedded in the ORF2 coding sequence; one encoded a putative protein with identifiable homology to SAT proteins of members of the genus Parvovirus.

Post-kala-azar dermal leishmaniasis as an immune reconstitution inflammatory syndrome in a patient with acquired immune deficiency syndrome

Post‐kala‐azar dermal leishmaniasis (PKDL) is a complication of visceral leishmaniasis (VL) observed mainly in Sudan and India where it follows treated VL in 50% and 10% of cases, respectively. We report a 46‐year‐old patient with acquired immune deficiency syndrome who, 7 months after diagnosis of VL, developed PKDL and uveal leishmaniasis following HAART‐induced immune recovery. In southern Europe PKDL seems to be an emerging clinical presentation among human immunodeficiency virus (HIV)‐infected patients experiencing HAART‐induced immune recovery after a previous diagnosis of VL. The best treatment among HIV‐infected patients remains to be determined.

Human parvovirus 4 in the bone marrow of Italian patients with AIDS.

Human parvovirus 4 (PARV4) is a recently discovered member of the Parvoviridae. We investigated the presence of this virus in bone-marrow aspirates of 35 Italian patients with AIDS. Viral DNA was detected by polymerase chain reaction in over 40% of patients (16/35). The infection was most prevalent in injection drug users (IDU; 12/18; 66.7%) as opposed to non-IDU (4/17; 23.5%). PARV4 infection is widespread in Italian patients with AIDS.

Liver-related factors associated with low vitamin D levels in HIV and HIV/HCV coinfected patients and comparison to general population.

OBJECTIVES Low 25-Hydroxyvitamin D (25[OH]D) was associated with severe fibrosis and low sustained virological response (SVR) after interferon (IFN)-based therapy in chronic hepatitis C. Furthermore, hypovitaminosis D was reported in HIV-infected individuals, but its role in liver disease progression in HIV/HCV coinfection is unknown. METHODS 25(OH)D was retrospectively measured in 237 HIV-infected patients (93 with HCV coinfection) and 76 healthy controls. Multivariate analysis included season, immuno-virological data, combined antiretroviral therapy (cART) and, in a subgroup of 51 HIV/HCV-genotype 1 coinfected patients, factors influencing SVR to pegylated-IFN and ribavirin. In a group of 20 patients, liver expression of cytochrome (CY)-P27A1 and CYP2R1, 25-hydroxylating enzymes, was assessed by immunohistochemistry. RESULTS Median 25(OH)D levels were 23.4 (interquartile range 16.7-33.7) ng/mL in the HIV-infected population and 24 ng/mL (18.3-29.5) in healthy controls (p=0.9). At multiple regression analysis, only winter/spring measurements correlated with lower 25(OH)D levels. No correlation with HCV coinfection, nor with cART regimens was found. Low 25(OH)D was independently associated with advanced fibrosis in HIV/HCV coinfected patients (p=0.023), whereas no association emerged with SVR to IFN-based therapy. CYP27A1 and CYP2R1 expression was associated neither with 25(OH)D serum levels nor with HCV-infection, liver histology, or cART. CONCLUSIONS In our experience, despite the high prevalence of 25(OH)D insufficiency, HIV and HCV-infection did not seem to influence vitamin D status. The role of HIV, HCV and cART on hypovitaminosis D needs further validation in larger cohorts that account for the vitamin levels in general populations and for seasonal and regional variability.

Persistent parvovirus B19-induced anemia in an HIV-infected patient under HAART. Case report and review of literature

Recent reports document resolution of human parvovirus B19-related pure red blood cell aplasia (PB19-PRCA) in HIV-infected patients upon commencement of highly active antiretroviral therapy (HAART). This article describes a patient with PB19-PRCA who, despite fully suppressive HAART, required cyclic administration of intravenous human immunoglobulin over a period of 17 months before PB19 seroconversion and subsequent resolution of relapsing severe anemia. All reports in the English literature describing PB19-related hematologic abnormalities in the post-HAART era are also described herein.

Louse-Borne Relapsing Fever (Borrelia recurrentis) in a Somali Refugee Arriving in Italy: A Re-emerging Infection in Europe?

Introduction Louse-borne relapsing fever (LBRF) is an acute febrile infection that is typically characterized by one to three fairly regular waves of bacteremia [1,2]. It is caused by Borrelia recurrentis, a motile spirochete that measures 5 to 40 μm in length. The microorganism is transmitted from person to person by the human body louse (Pediculus humanus humanus). Disruptions in sanitation during wartime and mass migrations of people provide conditions that favor the propagation of body lice and thus the occurrence of outbreaks of the disease [1,3]. LBRF is endemic in East Africa (e.g., Ethiopia, Eritrea, Somalia, and Sudan) with the highest number of cases observed in Ethiopia, where it is the seventh most common cause of hospital admission and the fifth most common cause of death [4,5]. We report here the first case of imported LBRF observed in Lombardy (northern Italy) in a Somali refugee.

Simultaneous onset of primary cutaneous B-cell lymphoma and human herpesvirus 8-associated Kaposi's sarcoma

We report the simultaneous occurrence of Kaposis sarcoma (KS) and primary cutaneous B‐cell lymphoma (CBCL) of the leg in a 79‐year‐old woman, seronegative for HIV‐1, HTLV‐1 and HTLV‐2. The CBCL underwent complete clinical remission after local radiotherapy, whilst the KS became disseminated within a year following diagnosis. However, 2 years after the diagnosis of KS, the patient died with neurological symptoms. These were presumed to be due to involvement of the central nervous system by lymphoma, although in the absence of an autopsy, this could not be proven. Skin biopsies from the original KS and CBCL lesions, as well as short‐term culture of spindle cells from the KS lesion and peripheral blood mononuclear cells (PBMC), were studied by semiquantitative polymerase chain reaction (PCR) using primers specific for DNA sequences of a novel γ‐herpesvirus‐8 (HHV‐8). PCR studies were strongly positive for the virus on KS cells and PBMC; conversely, a low viral load was found on CBCL cells. A high titre of serum IgG antibodies reacting with the nuclei of the HHV‐8 positive cell line BCP‐1 was found. These data suggest that reactivation of latent infection with HHV‐8 had occurred in this patient, and that HHV‐8 is directly involved in KS, but not in CBCL of the leg, an aggressive variant of CBCL.

Evidence for Kaposi's sarcoma-associated herpes virus infection in patients with idiopathic pulmonary arterial hypertension: a case series and review of the literature

ABSTRACT Background: The pathogenesis of idiopathic pulmonary arterial hypertension (IPAH) is unknown. Recent molecular and immunohistochemical evidence has demonstrated the presence of Kaposis sarcoma-associated herpes virus (KSHV) at high frequency in lung tissue from patients with IPAH, suggesting a possible role for this virus in the pathogenesis of the disease. Materials and methods: Eighty-seven patients with IPAH (n = 45) or other forms of pulmonary hypertension (n = 42) were prospectively assessed for serologic evidence of KSHV, Epstein–Barr virus (EBV) and human cytomegalovirus (HCMV) infection. Immunofluorescence assays specific for antibodies against latency-associated and lytic antigens of KSHV, as well as commercially available kits that detect antibodies against HCMV and EBV nuclear antigens, were employed. Results: Only one patient with IPAH (2.2%) and one of the patients with other forms of pulmonary hypertension tested seropositive for KSHV. In contrast, 100% and more than 90% of patients with both forms of pulmonary hypertension were positive for EBV and HCMV antibodies, respectively. Conclusions: Italian patients with IPAH do not exhibit serologic evidence of KSHV infection despite a normal ability to mount antibody-mediated responses toward human herpes viruses. KSHV is unlikely to play a role in the pathogenesis of IPAH.