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ObjectivesOur aim was to investigate the durability of different initial regimens in patients starting ART with CD4+ counts 5 log10 copies/mL.MethodsThis was a retrospective study of HIV-infected patients prospectively followed in the ICONA cohort. Those who started ART with boosted protease inhibitors (bPIs), NNRTIs or integrase strand transfer inhibitors (InSTIs), with CD4+ 5 log10 copies/mL, were included. The primary endpoint was treatment failure (TF), a composite endpoint defined as virological failure (VF, first of two consecutive HIV-RNA >50 copies/mL after 6 months of treatment), discontinuation of class of the anchor drug or death. Independent associations were investigated by Poisson regression analysis in a model including age, gender, mode of HIV transmission, CDC stage, HCV and HBV co-infection, pre-treatment HIV-RNA, CD4+ count and CD4+/CD8+ ratio, ongoing opportunistic disease, fibrosis FIB-4 index, estimated glomerular filtration rate, haemoglobin, platelets, neutrophils, calendar year of ART initiation, anchor drug class (treatment group) and nucleos(t)ide backbone.ResultsA total of 1195 patients fulfilled the inclusion criteria: 696 started ART with a bPI, 315 with an InSTI and 184 with an NNRTI. During 2759 person-years of follow up, 642 patients experienced TF. Starting ART with bPIs [adjusted incidence rate ratio (aIRR) (95% CI) 1.62 (1.29–2.03) versus starting with NNRTIs; P ConclusionsIn patients starting ART with 5 log10 HIV-RNA copies/mL, the durability of regimens based on InSTIs was longer than that of NNRTI- and bPI-based regimens.

To describe the clinical pattern of viral central nervous system (CNS) infections and compare meningitis and encephalitis. This is a retrospective study reporting the clinical characteristics and outcome of 138 cases of viral meningitis and meningoencephalitis in a real life experience at a referral centre in Turin, Northern Italy. Enteroviruses were predominant in younger patients who were mainly presenting with signs of meningitis, had shorter hospital admission and absence of complications, whereas herpesviruses had more often signs of encephalitis, were more frequent in elderly patients, had longer hospital admission and frequent complications and sequelae. Two main clinical entities with different epidemiology, clinical aspects and prognosis may be identified within the group of viral CNS inefctions.

To the Editor: Chikungunya virus (CHIKV) has become a global health problem. Clinical manifestations are not specific and are difficult to differentiate from those of similar viral diseases (e.g., dengue and Zika virus disease). Diagnostic laboratories must be prepared to meet the changing epidemiology of viral diseases. CHIKV infection is currently identified by viral genome detection, using reverse transcription PCR (RT-PCR), viral culture, and serologic testing for IgG and IgM by indirect immunofluorescence (IFA) or ELISA. RT-PCR is most sensitive during the early phase of CHIKV infection (within 5–7 days of symptom onset), but its use is limited by the short viremic phase of the disease. After the acute phase, serologic testing for IgG and IgM is a more accurate indicator of disease. Molecular and serologic tests are complementary, reliable, and sensitive methods, but they require special equipment and a medium-to-high level of technical skill that may not be available in many laboratories, especially those in rural areas, where outbreaks usually occur.

Acute hepatitis C virus (HCV) infection evolves to chronicity in 50-84% cases. Treatment with interferon-alpha (IFN-alpha) was repeatedly found to provide sustained cure rates higher than that in chronic HCV infection, but the optimal treatment strategy has not yet been defined. In a multicentre open-label study, we investigated the therapeutic performance of a short course of pegylated (peg) IFN-alpha in patients with acute HCV hepatitis. Peg IFN-alpha2b, 1.0-1.5 micro g/kg weekly, was administered for 12 weeks. Forty-six patients were enrolled; 26 of them were intravenous drug users. Eleven patients had jaundice. Treatment was started within 1-90 days from the peak alanine aminotransferase. Treatment was well tolerated with a single dropout (2%). Thirty-three of 46 patients (72%) had a sustained virological response (SVR) after a 6 months post-treatment follow-up, 8 (17%) relapsed after treatment and 4 were nonresponders (9%). A lower peak viraemia, receiving at least 1.2 micro g/kg of peg IFN-alpha, and a negative HCV-RNA at week 4 and week 12 were predictors of SVR. Thus, in patients with early (week 4) viral response, a short course of peg IFN-alpha at a weekly dose >1.2 micro g/kg, may be a valuable option for the treatment of acute HCV hepatitis.

Chikungunya virus (CHIKV) has recently emerged in the Caribbean. In Italy, CHIKV vector is documented in the Po river valley; therefore, a risk for autochthonous outbreaks is present. We report a case series of seven imported CHIKV infections in travelers returning from the Caribbean and Latin America occurring between June and November 2014, in the area of Turin, Northwest Italy, 3 years after the last imported cases were reported. These cases are a reminder of the need to always consider CHIKV infection in travelers from these epidemic areas as well as the importance of a prompt diagnosis.

Hantaviruses belong to the Bunyaviridae family, which is comprised of Bunyavirus, Phlebovirus, Nairovirus, and Hantavirus. Euroasiatic Hantaviruses belong to two distinct subfamilies: Murinae (comprising Hantaan, Dobrava, and Seoul viruses), which are responsible for hemorrhagic fever with renal syndrome (HFRS), and Arvicolinae (comprising Puumala virus), responsible for nephropathia epidemica (NE) and HFRS. On the contrary, the New World Hantavirus belongs to the Sigmodontinae subfamily and includes the North American viruses: Sin Nombre, Monongahela; New York, Bayou, Black Creek Canal, and the South American, which comprise the Andes, Oran, Lechiguanas, Laguna Negra, Juquitiba; both groups are responsible for the hantavirus pulmonary syndrome (HPS).

A constant surveillance of susceptibility to antimalarials allows to optimize prevention and treatment of malaria in nonendemic countries. In vitro susceptibility of imported Plasmodium falciparum isolates to chloroquine, quinine, mefloquine, halofantrin, pyronaridine, and amodiaquine was analyzed by WHO Micro-test Mark III; IC50 and IC90 were calculated by WHO Log-probit. Sixty-seven tests were performed. All the infections were acquired in Africa: 14.9% in East Africa and 85.1% in West Africa (WA). IC50 and IC90 (micromol/L) were chloroquine: 0.129 and 0.648; amodiaquine: 1.134 and 5.445; mefloquine: 0.38 and 0.868; quinine: 0.193 and 0.478; halofantrin: 3.27 and 25.35; pyronaridine: 11.504 and 51.996. Higher IC50 and IC90 were observed in East Africa versus West Africa strains. All strains were susceptible to quinine and mefloquine; chloroquine resistance, 14%; amodiaquine resistance, 33%, with cross-resistance to chloroquine (r = 0.93; P.0001); halofantrin resistance, 3.6%, no cross-resistance with chloroquine; low susceptibility to pyronaridine (66.7%), with cross-resistance with chloroquine (r = 0.38, P0.05). Lower levels of chloroquine resistance were observed in 2000-2003 as compared with prior data; thus, the reemergence of chloroquine susceptibility in Africa may be hypothesized.

Anisakis pathology is due mainly to two mechanisms: allergic reactions (from isolated urticaria and angioedema to life-threatening anaphylactic shock associated with gastrointestinal symptoms or ‘gastroallergic anisakiasis’), and direct tissue damage, due to invasion of the gut wall, development of eosinophilic granuloma, or perforation (gastric or intestinal anisakiasis). Anisakiasis is a misdiagnosed and underestimated cause of acute abdomen: most patients undergo laparotomy, and virtually no cases are diagnosed before surgery. In some cases, diagnosis is obtained accidentally during other pathologic investigations. We report a case of acute abdomen due to terminal ileum involvement. Microscopic examination of the resected segment showed the presence of helminthic sections consistent with larvae of Anisakis spp. A history of raw fish ingestion was recorded. Histopathologic features are illustrated. A short but up-to-date review of the literature on diagnostic devices (particularly imaging and serology), clinical aspects and therapy is presented.

In Italy the prevalence of recent HIV infection (RHI) isn't currently monitored. Early diagnosis is crucial to allow introduction of antiretroviral therapy (cART) in the recent phase of infection. We aimed to estimate the proportion and the determinants of RHI among patients enrolled in the ICONA cohort; we explored differences in the median time from HIV diagnosis to cART initiation and in the viro-immunological response between RHI and Less Recent HIV infections (NRHI). We included antiretroviral-naïve HIV-positive patients enrolled in the cohort with documented dates of HIV-negative and positive antibodies tests, grouped in RHI (estimated date of seroconversion within 12 months of enrolment) and NRHI. Proportion of RHI and the trend of this proportion by calendar period (1996-2014) were investigated (Chi-square test). Logistic regression analysis was employed to identify factors associated with RHI. The time from seroconversion to cART initiation was compared in RHI and NRHI overall and after stratification by calendar period (survival analysis). We finally explored the time from starting cART to HIV-RNA

Introduction Aim of the study was to evaluate possible disparities in access and/or risk of virological failure (VF) to the first antiretroviral (ART) regimen for migrants compared to Italian-born patients and to assess determinants of failure for the migrants living with HIV. Methods All native and migrant naïve patients enrolled in ICONA in 2004–2014 were included. Firstly, variables associated to ART initiation were analyzed. In a second analysis, the primary endpoint was time to failure after at least six months of ART, defined as: (a) VF (first of two consecutive viral load (VL) >50 and >200 copies/mL); (b) treatment discontinuation (TD) for any reason; and (c) treatment failure (TF: confirmed VL >200 cp/mL or TD). A Poisson multivariable analysis was performed to control for confounders. Results A total of 5777 HIV-pos ART-naïve patients (1179 migrants and 4598 natives) were evaluated. Most migrants were from sub-Saharan Africa (35.3%) and South-Central America/Caribbean (29%). Median duration of residency in Italy was five years (IQR 1–10). Baseline characteristics significantly differed between the two groups (Table 1); in particular, lower CD4 counts and higher frequency of AIDS events were observed in migrants vs natives. When adjusting for baseline confounders, migrants presented a lower chance to initiate ART compared to natives (OR 0.78, 95% CI 0.65–0.93, p=0.006). After ART initiation, the incidence rate of VF >50 cp/mL was 15.5 per 100 person-years (95% CI 12.8–18.8) in migrants and 8.9 in natives (95% CI 7.9–9.9), respectively. By multivariable analysis, migrants had a significantly higher risk of VF, both >50 cp/mL (OR 1.50, 95% CI 1.17–1.193, p=0.001) and >200 cp/mL (OR 1.59, 95% CI 1.23–2.05, p

Vaptans, vasopressin V2 receptor antagonists, are new drugs indicated in the treatment of Inappropriate ADH Secretion Syndrome (SIADH). We report a case of SIADH in a patient affected by AIDS. During a hospitalization for an ongoing CMV meningitis she developed a severe hyponatremia (114 mEq/L), which was initially treated with restriction of free water. When the diagnosis of SIADH was done (plasma Osmolarity 240 mOsm/kg, urinary sodium30 mEq/24 h, normal volemia, lack of hypocorticosurrenalism and hypothyroidism), tolvaptan was given at the dose of 15 mg/day. Important fluctuations of plasma Na were observed thereafter (119-143 mEq/L). The progressive reduction of the drug (to 2 mg/die) allowed a stable correction of natremia. Unconsciousness determined the impossibility of the patient to freely access to water, thus explaining the fluctuations of natremia and the difficult management of the drug. Only a posology reduction to doses lower than those available for sale allowed a stable correction of natremia.

By DNA sequence analysis we identified two new strain types and five novel sporadic variations among 25 isolates of Pneumocystis carinii f. sp. hominis obtained from 19 human immunodeficiency virus-positive patients. Of these, 13 were infected with a single strain and 6 were coinfected. Fifteen different combination types were identified among the 18 strains for which complete molecular typing was accomplished.

We describe seven cases of meningitis in a group of young Italian travelers coming back from India. Virologic studies identified echovirus-4 as the cause of this cluster of cases, the first imported echovirus outbreak in Italy. Enteroviruses may play an important role in undiagnosed fevers in travelers.

The opportunistic pathogen Pneumocystis carinii (PC) is a frequent cause of a life-threatening pneumonia in human immunodeficiency virus (HIV)-infected individuals and in other immunocompromised hosts. Specimens obtained from 128 bronchoalveolar lavage (BAL) fluid samples from 123 HIV-positive patients with pulmonary disease and undergoing a diagnostic bronchoscopy were evaluated to detect this organism. We have developed a rapid DNA extraction procedure for nested polymerase chain reaction (PCR) using two sets of primers (pAZ102-E, pAZ102-H and P1 = 5'-CTAGGATATAGCTGGTTTTC-3' and P2 = 5'-TCGACTATCTAGCTTATCGC-3'). The results were compared using cytological techniques (direct wet mount, Giemsa, toluidine blue O) and related to the clinical follow-up of patients. The nested PCR had a 91% sensitivity and a 93% specificity. The effect of chemoprophylaxis and the evaluation of the follow-up of patients are discussed. Nested PCR may represent an important additional tool, along with current cytological methods, for the detection of P. carinii; however, at present it cannot replace routine microbiological methods more simple and less expensive.

Detection of parasites in culture or by microscopy is still necessary to make diagnosis of visceral leishmaniasis (VL). Serological methods still need assessment, as they are quick but not very sensitive, especially in immunosuppressed subjects. This paper compares the results obtained with three serological methods (indirect immunofluorescence test (IFAT), direct agglutination test (DAT), and enzyme-linked immunosorbent assay (ELISA) and the specific cell-mediated immune response, evaluated as proliferation and IFN-γ production by peripheral blood lymphocytes (PBL) following stimulation with heat-killed L. infantum promastigotes. PBL and sera were obtained from 10 healthy donors, 3 VL patients in acute phase, and 3 patients recovering after two glucantim treatment courses. No false positive results were observed with the serological methods. IFAT can be considered the most sensitive and best suited for follow-up, as it allowed a good discrimination between the acute and remission phase. DAT did not discriminate between healthy donors and remission-phase patients, whereas ELISA is unsuited for follow-up, as it did not show any significant difference between remission- and acutephase patients. Assessment of the cellular response is not recommended for making a diagnosis, because false positive results are frequent. However, a strong cellular response in a patient stands for a successful treatment. IFN-γ titration is preferable to the proliferation test, because it gives earlier results and does not require the use of radioactive isotopes.

Dynamics of human immunodeficiency virus type 1 (HIV-1) tropism after antiretroviral therapy (ART) initiation and their association with disease progression are poorly investigated. This was a cohort study on subjects from the ICONA cohort receiving ART with persistently detectable (PD) or persistently undetectable (PU) viral load (VL) and with stored plasma or peripheral blood mononuclear cell (PBMC) samples at 2 time-points (T1, T2) after ART initiation. HIV-1 co-receptor tropism was determined by V3-loop sequencing and the geno2pheno algorithm. A switch in viral tropism was defined if the tropism classification at T2 differed from that observed at T1. Time to disease progression, defined as the occurrence of a new acquired immune deficiency syndrome (AIDS)-defining event/death from T2, was also evaluated. One hundred ninety-five patients were analyzed (124 PD, 71 PU). Over a median follow-up of 22.6 (19.8–28.1) months, PD and PU patients showed similar rates (95% confidence interval) of switch to a non-R5 virus [PD: 6.9 (3.7–11.2)/100-person-years of follow-up (PYFU); PU: 8.0 (3.4–14.5)/100-PYFU; P = 0.63] and of switch to a R5 virus [PD: 15.4 (7.3–26.4)/100-PYFU; PU: 8.1 (2.5–16.7)/100-PYFU; P = 0.38]. Switch to non-R5 virus was predicted by nadir CD4+ before T1. Twenty-two (18%) PD and 4 (6%) PU subjects experienced disease progression (P = 0.02). The risk of disease progression was independently associated with a switch in co-receptor tropism (adjusted hazard ratio = 4.06, 95% CI: 1.20–13.80, P = 0.03) as well as age, AIDS diagnosis, nadir CD4+ before T2, current CD4+, and VL. Switch of HIV-1 tropism under ART occurs in both directions, with similar rates in subjects with PD or PU VL and it might be predictive of future unfavorable clinical outcome.

Introduction : Criteria of cART initiation after a first ADE have been modified over time based on evidence suggesting that treatment should be initiated earlier. The impact of these changes on clinical practice is unknown. Objective of this analysis was to evaluate temporal changes of time of starting cART after a first diagnosis of ADE in ART-naive patients (pts). Methods : All HIV+ enrolled in ICONA Foundation Study who presented with a diagnosis of ADE while cART-naive regardless of CD4 cell count were included. Pts were grouped according to have ADE for which additional medications that may have interactions with cART are required (Tb, atypical mycobacteriosis, non-Hodgkin lymphoma) [group A], ADE treatable only by cART (PML, isosporidiasis/cryptosporidiasis, KS, wasting syndrome) [group B] and ADE treatable with specific drugs (PCP, toxoplasmic encephalitis, CMV disease, esoph candidiasis, bacterial pneumonia, cervical cancer, cryptococcosis) [group C]. Standard survival analysis by KM was used to estimate the cumulative percentage of pts starting cART, overall and after stratification for calendar period of diagnosis (1996-2000, 2001-2008, 2009-2011) and type of ADE (groups A, B, C). Multivariable Cox regression was used to investigate association between calendar year of ADE and time to cART initiation after controlling for demographics. Summary of results : A total of 715 pts with a first ADE were observed over 1996-2011 (group A, n=187; B, n=123; C, n=405). 519 (73%) male, median age 38 (IQR:33-45), median CD4+ 64 (23-187)/mm 3 and HIV/RNA 5.25 (4.57-5.70) log 10 cps/mL, with no differences by calendar period. By 30 days from ADE, 23% (95% CI: 19-27) of those diagnosed in 1996-2000 have started cART vs. 32% (95% CI: 25-39) in 2001-2008 and 36% (28-44) after 2009 (log-rank p=0.001). After stratifying by CD4 at ADE, 45% of pts with CD4 201/mm 3 had started cART by 30 days (p

We describe seven cases of meningitis in a group of young Italian travelers coming back from India. Virologic studies identified echovirus-4 as the cause of this cluster of cases, the first imported echovirus outbreak in Italy. Enteroviruses may play an important role in undiagnosed fevers in travelers.

The emergence of Plasmodium falciparum drug-resistance, especially chloroquine resistance, represents one of the main obstacles to the control of malaria. Several studies have shown that in P. falciparum the mechanism of chloroquine resistance is linked to specific point mutations in the pfcrt gene of the parasite. In the present study we have analyzed 120 Italian imported malaria cases to evaluate the prevalence of 76T and 220S mutantions in the pfcrt gene. Moreover, the correlation between the presence of pfcrt point mutations and in vitro chloroquine resistance has been evaluated on 25 plasmodial isolates. The results showed a high prevalence of the pfcrt point mutations in isolates analyzed and a significant association between point mutations and in vitro chloroquine resistance. Molecular screening on imported malaria cases can be a useful tool to be employed in surveillance activity and also in monitoring the development and spread of drug resistance in endemic areas.

The development in Plasmodium falciparum of the resistance to chloroquine (CQ) constitutes a public health priority, due to its direct influence in childhood mortality. The molecular basis for CQ resistance (CQR) is still unclear but, recently, a new relevant gene, named pfcrt, with several point mutations was identified in P. falciparum. Two mutations, K76T and A220S, have been considered crucial for CQR in further studies, making the pfcrt a good candidate as determinant for CQR in P. falciparum. To contribute to this topic, we have undertaken a molecular screening on 164 P. falciparum isolates from Africa: 120 isolates were Italian imported malaria cases, 27 and 17 isolates were from a school-children survey from Congo and Tanzania, respectively. In vitro tests (pLDH and WHO-Mark III tests) for CQ sensitivity have been also carried out on 28 plasmodial isolates and results compared to those obtained by molecular analysis in the same isolates. The SVIET pfcrt haplotype has been identified in the samples from Congo, and this is the first time that this haplotype is detected in Africa. Our results give further evidence to the reliability of the 76T (and the linked 74I-75E) pfcrt point mutation as molecular marker for CQR.

Cyclospora sp, a recently described protozoan, is associated with prolonged self-limiting and relapsing diarrhea. The species has a worldwide distribution and a high prevalence in tropical countries. Some reports suggest that the agent is a common cause of travelers' diarrhea.

Sir, Intravenous artesunate is the treatment of choice for severe and complicated malaria, according to the WHO 2010 guidelines, after large clinical trials in Africa (AQUAMAT) and South-East Asia (SEAQUAMAT) demonstrated a substantial benefit in terms of survival when this drug was compared with quinine; artesunate also proved to be very well tolerated. We report the case of a 22-year-old Italian woman who presented to Amedeo di Savoia Hospital in Turin in September 2011, with fever, chills and headache after a 4 week stay in Sierra Leone, without taking malaria chemoprophylaxis. Her physical examination showed a temperature of 408C, dehydration and splenomegaly. Blood tests showed a normal leucocyte count, haemoglobin 14.4 g/dL, thrombocyte count 32000/mm, lactate dehydrogenase (LDH) 2722 U/L and total bilirubin 5.7 mg/dL. Peripheral thick and thin blood films showed Plasmodium falciparum trophozoites (3.8% parasitized red blood cells). A treatment with oral artemether/lumefantrine was started. The following day, the parasitaemia was 6.9%; since the parasitaemiawas increasing and the patient was fulfilling WHO 2010 criteria for severe malaria (hyperparasitaemia and jaundice), intravenous artesunate (Guilin Pharmaceutical Co., Shanghai, China) dosed at 2.4 mg/kg every 12 h was started. After 6 h, the parasitaemia had dropped to 2% and was 0.01% after 24 h. After 36 h, the patient was switched to oral artemether/lumefantrine. On day 4, parasitaemia was absent and LDH was 1449 U/L. On day 8 she had another febrile peak and laboratory signs of haemolytic anaemia (haemoglobin 8.1 g/dL, LDH 2406 U/L, bilirubin 3.1 mg/dL, reticulocytes 150/1000 and haptoglobin ,2 mg/dL). No parasites were found on the blood smear. Immunohaematological haemolysis and glucose-6-phosphate dehydrogenase deficiency were ruled out by a cold agglutinins test, Coombs test, autoantibodies and glucose-6-phosphate dehydrogenase test. On day 14 (haemoglobin 5.6 g/dL), a blood transfusion was prescribed. In the following days the patient’s condition improved and she was discharged on day 18. On day 40, the patient was asymptomatic, thin and thick blood films were negative, and haematological and biochemical parameters were within the normal range. The patient gave written informed consent for the publication of these data. Haemolysis is not reported as a side effect of artesunate treatment in the cited trials (only some expected cases of blackwater fever occurred immediately after treatment). Nonetheless, subclinical haemolysis could be undiagnosed in such settings. Zoller et al. recently published a case series in European travellers with artesunate-treated imported malaria, including six cases with haemolysis occurring 14–31 days and resolving 3–6 weeks after treatment. The event was more frequent when high doses were used. In the reported case, an LDH flare and decrease in haemoglobin reappeared after parasite and fever clearance, and were associated with fever (37.5–388C) and conjunctival jaundice, indicating a haemolytic syndrome. A direct drug toxicity is unlikely, given the short half-life of the molecule (time to final detection: 2 h); although, due to the drug’s production outside European standards of good manufacturing practice, the presence of contaminants cannot be excluded. A further possibility is haemolysis due to lumefantrine, which has a longer half-life; however, this has never been reported, despite the long-term worldwide use of artemether/lumefantrine. Overall, a clear explanation for the haemolysis in this reported case is lacking. Since unexplained haemolysis has been repeatedly observed, specific surveillance in artesunate-treated patients is required.

Imported malaria has been an important public health problem in Western countries in the last 20 years, since international travel has become an increasing habit for nonimmune populations and since chemoresistance to most antimalarial drugs has been spreading throughout the world. Moreover, immigration from African and Asian countries has been rapidly increasing, especially in Italy in the last few years. Malaria had been widespread in Italy in the past, but no new autochthonous cases have been reported since 1961. Nonetheless the number of reported cases throughout the country has been steadily growing because of imported malaria1-3 in nonimmune travelers as well as in immigrants from tropical countries. In our experience as well as according to other statistics, the vast majority of patients have Plasmodium falciparum malaria acquired in Africa.4,5 The clinical spectrum of this disease is wide, and severe cases are frequently observed, including a few fatal cases, which, although rare, are highlighted by mass media and impress the public opinion.5-8 The purpose of this study was to examine the clinical spectrum of malaria, with particular interest in severe falciparum malaria, and to define the frequency of this phenomenon and epidemiologic characteristics of patients who experience it as a life-threatening disease.

Malaria diagnosis relies on observation of parasites in blood smears and the Giemsa-stained thick blood smear (G-TS) is the reference test. Diagnosis by G-TS in low-density infections requires long periods of observation and experienced microscopists. Examination of Giemsa-stained thin smears enables more reliable differentiation of species but may miss low-grade infections. Fluorescent stains may offer an alternative technique. We compared the Giemsa technique with 4,6-diamidine-2-phenilindolo-propidium iodide (DAPI-PI) stainings in order to evaluate the time required for diagnosis. A Plasmodium falciparum-infected blood specimen was diluted to obtain concentrations ranging from 6192 to 24 parasites/microliters (p/microliter), and thin and thick smears were stained with the two methods. The DAPI-PI proved useful: parasites were easily recognized and their morphology was preserved in thin and thick smears. The method allowed more rapid evaluation of thin smears as compared with Giemsa staining and enabled recognition of parasites in case of low-level parasitemias. The DAPI-PI staining technique may acquire an important role in malaria diagnosis, especially in nonendemic countries where technicians are not experienced with G-TS; in developing countries, it could be used in epidemiologic surveys of populations with low-density parasitemias, for which it enables a fast examination of smears and possibly the identification of parasite species.

OBJECTIVE: We evaluated the prevalence of HIV-1 non-clade B over time in a formerly clade B-restricted area. Protease and reverse transcriptase regions of the pol gene were used for phylogenetic and recombination analysis and for clade assignment to HIV-1 A-D, F-H, J, and K strains of the M group. METHODS: The pol gene of 349 HIV-1 patients belonging to the Italian Cohort Naive for Antiretrovirals (ICONA) were genotypically analyzed to study the prevalence of antiretroviral-associated resistance mutations. All HIV-1 pol sequences and 32 HIV reference strains were analyzed, including the reference strains for the major HIV-1 subtypes. The non-clade B sequences according to the HIV-1 Subtyping Tool program were further studied by a bootscan analysis (SimPlot) to investigate the likelihood of recombination between subtypes. RESULTS: Phylogenetic analysis detected 19 of 349 (5.4%) non-clade B subtypes. The proportions of patients carrying non-clade B virus before and after 1997 were 1.9% and 8.4%, respectively (p =.008). Among whites, heterosexual infection and female gender were significantly associated with the presence of non-clade B subtypes (p =.001 and.005, respectively). Non-clade B HIV-1 was harbored by 14.5% of the heterosexuals who were found to be HIV-1 positive after 1997, 60% of whom were women. Bootscan analysis identified four strains as F, two as A, one as C, one as G, and 11 (57.9 %) as non-clade B recombinant subtypes. CONCLUSION: Detection of HIV-1 subtypes and intersubtype recombinants in a previously clade B-homogeneous area indicates that the HIV-1 epidemic is evolving in Italy and that heterosexuals and women are at increased risk of infection with non-clade B HIV-1 subtypes. Sequences inferred from the pol gene yield to establish the subtype of circulating HIV-1 strains. As a consequence, genotyping of pol gene for testing resistance to antiretrovirals warrants concomitant surveillance of non-clade B subtypes. ispartof: Journal of Acquired Immune Deficiency Syndromes vol:27 issue:5 pages:499-505 ispartof: location:United States status: published