Five cases of Plasmodium vivax malaria treated with artemisinin derivatives: the advantages of a unified approach to treatment.

OBJECTIVES: In endemic countries with a high level of chloroquine resistance, Plasmodium vivax malaria is associated with high morbidity and mortality. In these areas, the dihydroartemisinin-piperaquine combination resulted in clinical response, a more rapid clearance of parasitaemia, compared to chloroquine therapies, and reduction of recrudescence or reinfection. METHODS: We describe five cases of Plasmodium vivax malaria in returning travelers treated with dihydroartemisinin-piperaquine. RESULTS: All patients showed the early parasite clearance and no side effects. Our preliminary results suggest that the dihydroartemisinin-piperaquine combination is effective and safe even in imported cases. CONCLUSIONS: A unified treatment policy using the artemisinin combination therapy should be adopted even in non-endemic countries and larger studies are underway to support this strategy.

Severe Plasmodium ovale malaria complicated by acute respiratory distress syndrome in a young Caucasian man.

BACKGROUND: Although Plasmodium ovale is considered the cause of only mild malaria, a case of severe malaria due to P. ovale with acute respiratory distress syndrome is reported. CASE PRESENTATION: A 37-year old Caucasian man returning home from Angola was admitted for ovale malaria to the National Institute for Infectious Diseases Lazzaro Spallanzani in Rome, Italy. Two days after initiation of oral chloroquine treatment, an acute respiratory distress syndrome was diagnosed through chest X-ray and chest CT scan with intravenous contrast. Intravenous artesunate and oral doxycycline were started and he made a full recovery. CONCLUSION: Ovale malaria is usually considered a tropical infectious disease associated with low morbidity and mortality. However, severe disease and death have occasionally been reported. In this case clinical failure of oral chloroquine treatment with clinical progression towards acute respiratory distress syndrome is described.

Inguinal and anorectal Lymphogranuloma Venereum: a case series from a sexually transmitted disease center in Rome, Italy.

BACKGROUND: Lymphogranuloma venereum (LGV) is a sexually transmitted infection caused by L1, L2, L3 serovars of C. trachomatis (CT). Since 2003, LGV cases have been increasing in Europe. Aim of this report is to describe the LGV cases diagnosed in the largest STI center in Rome, Italy, from 2000 to 2016. This report shows that two clinically and epidemiologically different series of cases exist, and that, at present, the ano-rectal LGV represents the clinical variant occurring more frequently among men having sex with men (MSM), particularly those HIV-infected. CASE PRESENTATION: Ten cases of LGV were observed. Three were diagnosed in 2009 in HIV-negative heterosexuals patients that presented the classical genito-ulcerative form with lymphadenopathy. Seven cases were observed in 2015-2016 in HIV-infected MSM, that presented the rectal variant and L2b serovar infection; 4 of these had been misclassified as a chronic bowel disease. Chlamydia infection was confirmed by CT-specific PCR (ompA gene nested PCR), followed by sequence analysis to identify the serovar. All the patients were treated with doxycycline for 3 weeks, obtaining a complete response with healing of both clinical symptoms and dermatological lesions. CONCLUSIONS: Our findings suggest that, in case of persistent rectal symptoms in HIV-infected MSM, LGV should be taken into account and investigated through molecular analyses, in order to achieve a correct diagnosis and management of the patients.

Comparison of Leishmania typing results obtained from 16 European clinical laboratories in 2014.

Leishmaniasis is endemic in southern Europe, and in other European countries cases are diagnosed in travellers who have visited affected areas both within the continent and beyond. Prompt and accurate diagnosis poses a challenge in clinical practice in Europe. Different methods exist for identification of the infecting Leishmania species. Sixteen clinical laboratories in 10 European countries, plus Israel and Turkey, conducted a study to assess their genotyping performance. DNA from 21 promastigote cultures of 13 species was analysed blindly by the routinely used typing method. Five different molecular targets were used, which were analysed with PCR-based methods. Different levels of identification were achieved, and either the Leishmania subgenus, species complex, or actual species were reported. The overall error rate of strains placed in the wrong complex or species was 8.5%. Various reasons for incorrect typing were identified. The study shows there is considerable room for improvement and standardisation of Leishmania typing. The use of well validated standard operating procedures is recommended, covering testing, interpretation, and reporting guidelines. Application of the internal transcribed spacer 1 of the rDNA array should be restricted to Old World samples, while the heat-shock protein 70 gene and the mini-exon can be applied globally.

Left thigh phlegmon caused by Nocardia farcinica identified by 16S rRNA sequencing in a patient with leprosy: a case report.

BACKGROUND: In recent years, Nocardia farcinica has been reported to be an increasingly frequent cause of localized and disseminated infections in the immunocompromised patient. However, recent literature is limited. We report a case of left thigh phlegmon caused by N. farcinica that occurred in a patient with leprosy undergoing treatment with prednisone for leprosy reaction. CASE PRESENTATION: We describe the case of left thigh phlegmon caused by Nocardia farcinica in a 54-year-old Italian man affected by multi-bacillary leprosy. The patient had worked in South America for 11 years. Seven months after his return to Italy, he was diagnosed with leprosy and started multi-drug antibiotic therapy plus thalidomide and steroids. Then, during therapy with rifampicin monthly, minocycline 100 mg daily, moxifloxacin 400 mg daily, and prednisone (the latter to treat type 2 leprosy reaction), the patient complained of high fever associated with erythema, swelling, and pain in the left thigh. Therefore, he was admitted to our hospital with the clinical suspicion of cellulitis. Ultrasound examination and Magnetic Resonance Imaging showed left thigh phlegmon. He was treated with drainage and antibiotic therapy (meropenem and vancomycin replaced by daptomycin). The responsible organism, Nocardia farcinica, was identified by 16S rRNA sequencing in the purulent fluid taken out by aspiration. The patient continued treatment with intravenous trimethoprim/sulfamethoxazole and imipenem followed by oral trimethoprim/sulfamethoxazole and moxifloxacin. A whole-body computed tomography did not reveal dissemination to other organs like the lung or brain.The patient was discharged after complete remission. Oral therapy with trimethoprim/sulfamethoxazole, moxifloxacin, rifampicin monthly, clofazimine and thalidomide was prescribed to be taken at home. One month after discharge from the hospital the patient is in good clinical condition with complete resolution of the phlegmon. CONCLUSION: N. farcinica is a rare infectious agent that mainly affects immunocompromised patients. Presentation of phlegmon only without disseminated infection is unusual, even in these kinds of patients. In any case, a higher index of suspicion is needed, as diagnosis can easily be missed due to the absence of characteristic symptoms and the several difficulties usually encountered in identifying the pathogen.

Haemolytic anaemia in an HIV-infected patient with severe falciparum malaria after treatment with oral artemether-lumefantrine.

Intravenous (i.v.) artesunate is now the recommended first-line treatment of severe falciparum malaria in adults and children by WHO guidelines. Nevertheless, several cases of haemolytic anaemia due to i.v. artesunate treatment have been reported. This paper describes the case of an HIV-infected patient with severe falciparum malaria who was diagnosed with haemolytic anaemia after treatment with oral artemether-lumefantrine.The patient presented with fever, headache, and arthromyalgia after returning from Central African Republic where he had been working. The blood examination revealed acute renal failure, thrombocytopaenia and hypoxia. Blood for malaria parasites indicated hyperparasitaemia (6%) and Plasmodium falciparum infection was confirmed by nested-PCR. Severe malaria according to the laboratory WHO criteria was diagnosed. A treatment with quinine and doxycycline for the first 12 hours was initially administered, followed by arthemeter/lumefantrine (Riamet(®)) for a further three days. At day 10, a diagnosis of severe haemolytic anaemia was made (Hb 6.9 g/dl, LDH 2071 U/l). Hereditary and autoimmune disorders and other infections were excluded through bone marrow aspiration, total body TC scan and a wide panel of molecular and serologic assays. The patient was treated by transfusion of six units of packed blood red cell. He was discharged after complete remission at day 25. At present, the patient is in a good clinical condition and there is no evidence of haemolytic anaemia recurrence.This is the first report of haemolytic anaemia probably associated with oral artemether/lumefantrine. Further research is warranted to better define the adverse events occurring during combination therapy with artemisinin derivatives.

Clostridium difficile 027 infection in Central Italy.

BACKGROUND: Clostridium difficile (CD) has increasingly become recognised as a significant international health burden, often associated with the healthcare environment. The upsurge in incidence of CD coincided with the emergence of a hypervirulent strain of CD characterized as 027. In 2010, 8 cases of CD 027 infections were identified in Italy. Since then, no further reports have been published. We describe 10 new cases of CD 027 infection occurring in Italy. METHODS: Since December 2010, stool samples of patients with severe diarrhea and clinical suspicion of the presence of a hypervirulent strain, were tested for CD 027 by the Xpert C. difficile PCR assay (Cepheid, Sunnyvale, CA). Clinical, epidemiological and laboratory data were collected. RESULTS: From December 2010 to April 2012, 24 faecal samples from 19 patients who fit the above criteria were submitted to our laboratory. Samples were collected from 7 different hospitals.Of these, 17 had a positive PCR for CD and 10 were the epidemic 027 strain (59%). All PCR positive samples had a positive EIA toxin A/B test. Nine of 10 patients were recently exposed to antimicrobials and were healthcare-associated, including 4 with a history of long term care facility (LTCF) admission; the remaining case was community-associated, namely the wife of a patient with hospital-acquired CD 027 infection. Five patients experienced at least one recurrence of CD associated diarrhea (CDAD) with a total of 12 relapsing episodes. Of these, two patients had 5 and 6 relapses respectively.We compared the 10 patients with 027 CDAD versus the 7 patients with non-027 CDAD. None of the 7 patients with non-027 CDAD had a recent history of LTCF admission and no subsequent relapses were observed (p = 0.04). CONCLUSIONS: Our study shows that CD 027 is emerging in healthcare facilities in Italy. Whilst nosocomial acquisition accounted for the majority of such cases, 4 patients had history of a recent stay in a LTCF. We highlight the substantial risks of this highly transmissible organism in such environments. Moreover, 50% of our patients with CDAD from the 027 strain had high relapse rates which may serve to further establish this strain within the Italian health and social care systems.

Granulomatous myocarditis caused by Candida albicans in a canary (Serinus canaria).

Candida albicans is among the major agents of mucous membrane mycosis in humans and animals, with systemic and deep infections observed in immunocompromised hosts. We describe a case of fatal granulomatous myocarditis caused by C albicans in a 20-day-old canary (Serinus canaria). The etiologic diagnosis was confirmed by identifying characteristic morphologic features of the organism, combined with histochemical staining, and followed by the use of ad hoc biomolecular analysis.

Meningitis with cranial polyneuritis and cavernous sinus thrombosis by Borrelia crocidurae: First autochthonous case in Europe.

Borrelia crocidurae is endemic in West Africa, where it represents the leading cause of tick-borne relapsing fever (TBRF). TBRF typically presents with high fever and systemic symptoms, followed by recurrent episodes. Neurological complications may occur during febrile relapses. B. crocidurae is considered the most neurotropic agent of TBRF and is associated to severe neurological manifestations i.e. meningitis and encephalitis. To date, European cases of B. crocidurae infection have been reported in travelers returning from endemic areas. We report the first autochthonous case in Europe of B. crocidurae infection, presenting as meningitis with cranial polyneuritis and cavernous sinus thrombosis that were not preceded by classic febrile recurrences.

Molecular diagnosis of fatal Nocardia farcinica pneumonia in an HIV-negative patient.

Nocardiosis is a rare and potentially life-threatening infection caused by several species of the Nocardia genus. Most cases occur in immunocompromised patients, and a delay in establishing the diagnosis is common due to the non-specific clinical presentations and the difficulty in cultivating Nocardia. Although the majority of pulmonary nocardiosis cases are caused by Nocardia asteroides, cases of human infection due to N. farcinica are increasingly diagnosed due to recent developments in taxonomy and diagnostic methods. N. farcinica is a separate species from N. asteroides and appears to be more virulent and resistant to antibiotics. Herein, we describe the case of a 65-year-old HIV-negative immunocompromised patient with a fulminant bilateral pulmonary nocardiosis while on empirical treatment with trimethoprim/sulfamethoxazole and imipenem. Post-mortem diagnosis of N. farcinica infection was performed by means of DNA amplification and sequencing of the 65-kDa bacterial heat shock protein.

Plasmodium falciparum multiple infections, disease severity and host characteristics in malaria affected travellers returning from Africa.

BACKGROUND: The pathogenesis of malaria is the result of complex interactions between parasites, host and environment. Several studies have assessed the role of genetic characteristics of Plasmodium falciparum infection in the clinical severity of malaria infection comparing different genotypic determinants in mild and severe cases. The genes encoding the polymorphic merozoite surface proteins 1 (msp-1) and 2 (msp-2) and the dihydrofolate reductase (dhfr) of malaria parasites have been extensively used as markers to investigate the genetic diversity and the population structure of P. falciparum. The aim of this study was to assess the epidemiological, clinical, host- and parasite-related determinant factor of the genetic diversity of P. falciparum infections in travellers returning to Italy. METHODS: Between 1998 and 2001, we have retrospectively studied 64 inpatients all returning from African malaria-endemic countries. Designation of severe malaria was determined by using the World Health Organization (WHO) definition. P. falciparum infections detected by species-specific PCR were genotyped at the msp-1 and msp-2 loci and clones were determined. PCR and enzyme-digestion methods were used to screen the mutation occurring at codon 108. RESULTS: Multiple P. falciparum genotypes were detected in 32 patients (50%). The number of genotypes was correlated to different host characteristics. No association was found between allelic number of msp-1 or msp-2 and season of travel, absence of antimalarial prophylaxis, length of stay or blood parasitemia. At multiple analysis adjusted for few confounding variables, two variables showed a significant association with multiplicity of P. falciparum genotypes: male gender (p=0.018) and severity of disease (p=0.044). CONCLUSION: In our study all but one patients with severe malaria had a infection with a multiplicity of P. falciparum clones. At multivariate analysis the male gender, and the occurrence of severe malaria were significantly more commonly detected in patients affected by imported malaria with multiple clones.

Ceftaroline Plus Ampicillin Against Gram-Positive Organisms: Results from E-Test Synergy Assays.

In an era of increasing drug resistance and limited numbers of antimicrobials in the drug production pipeline, healthcare-associated infections represent a growing public health threat. When therapeutic options are limited, clinicians often resort to using antimicrobial combinations that produce a synergistic effect on the target pathogen. Novel antibiotics are therefore welcome in the daily practice of medicine. For example, ceftaroline is a broad-spectrum cephalosporin active against a variety of bacteria, including methicillin-resistant Staphylococcus aureus, but with limited activity against enterococci, particularly Enterococcus faecium. In this study, we tested the efficacy of ceftaroline against clinical isolates of gram-positive bacteria (S. aureus, Enterococcus faecalis, and E. faecium) by the broth microdilution and E-test assays, and then evaluated the synergistic effect of ceftaroline and ampicillin using the E-test method. The time-kill assay was used to confirm the data on selected strains. This drug combination has been recently shown to be effective against E. faecalis and could offer the advantage of cost-effectiveness (compared to other synergistic associations) as well as good tolerability. The E-test was chosen because of its relative simplicity of use that makes it suitable for routine clinical laboratories as a quick tool to guide clinicians when confronted with difficult-to-treat infections that may require an empirical approach. Our results indicate the presence of a synergistic effect of ceftaroline and ampicillin on most of the strains used, especially E. faecium and E. faecalis. The fact that two of those Enterococcus strains were vancomycin resistant suggests that the possible use of this combination for combating the spread of vancomycin-resistant enterococci should be explored.

Evaluation of the inactivation effect of Triton X-100 on Ebola virus infectivity.

BACKGROUND: The recent Ebola virus disease outbreak occurred in West Africa since December 2013 highlighted the need of appropriate virus inactivation procedures to be set up to allow the necessary processing of specimens outside BSL-4 facilities and to perform laboratory tests without affecting clinical decisions. For this purpose, international guidelines suggest the pre-treatment of the samples with Triton X-100. OBJECTIVES: Due to the limited scientific evidence about the efficacy of Triton X-100 on enveloped-viruses, the aim of this work was to evaluate the effect of Triton X-100 on the virus infectivity and to establish the optimal conditions for its use. STUDY DESIGN: We evaluated the effect of Triton X-100 on the infectivity of enveloped-viruses such as West Nile virus (WNV) and Ebola virus (EBOV) at different experimental conditions. The residual virus infectivity was measured by limiting dilution assay on Vero E6 cells. Repeated experiments were performed, as specified, and for the titration of residual infectivity each dilution was tested in triplicate. RESULTS: Results obtained with WNV showed that infectivity was reduced by 6 Logs, after 1h of treatment with different concentrations of Triton X-100 (ranging from 0.5% to 0.05%). This effect was not time-dependent using 0.1% Triton X-100. Subsequently, we applied the method on EBOV and one hour exposure to 0.1% Triton X-100 strongly affected EBOV infectivity (4 Logs of infectivity reduction). CONCLUSIONS: We report that Triton X-100, when used alone, is able to strongly reduce the infectivity of a classical enveloped virus such as WNV and we provide, for the first time, scientific evidence that 0.1% Triton X-100 efficaciously affect Ebola virus infectivity. Even though a complete virus inactivation is not achieved, Triton X-100 certainly can contribute to mitigate the risk for the workers of accidental infection and improve the overall safety of the laboratory procedures. Further studies must be performed to deeply investigate alternative solutions able to balance higher level of safety and good performance in clinical chemistry and hematology parameters analysis, necessary for the appropriate and effective management of EVD patients.

Immunological diagnosis as an adjunctive tool for an early diagnosis of tuberculous meningitis of an immune competent child in a low tuberculosis endemic country: a case report.

BACKGROUND: Pediatric tuberculous meningitis is a highly morbid, often fatal disease. Its prompt diagnosis and treatment saves lives, in fact delays in the initiation of therapy have been associated with high mortality rates. CASE PRESENTATION: This is a case of an Italian child who was diagnosed with tuberculous meningitis after a history of a month of headache, fatigue and weight loss. Cerebrospinal fluid analysis revealed a lymphocytic pleocytosis with predominance and decreased glucose concentration. Microscopy and conventional diagnostic tests to identify Mycobacterium tuberculosis were negative, while a non classical method based on intracellular cytokine flow cytometry response of CD4 cells in cerebral spinal fluid helped us to address the diagnosis, that was subsequently confirmed by a nested polymerase chain reaction amplifying a 123 base pair fragment of the M. tuberculosis DNA. CONCLUSIONS: We diagnosed tuberculous meningitis at an early stage through an innovative immunological approach, supported by a nested polymerase chain reaction for detection of M. tuberculosis DNA. An early diagnosis is required in order to promptly initiate a therapy and to increase the patient's survival.

Prevalence of pfcrt point mutations and level of chloroquine resistance in Plasmodium falciparum isolates from Africa.

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.

Leishmania infantum leishmaniasis in corticosteroid--treated patients.

BACKGROUND: The number of leishmaniasis cases associated with immunosuppression has increased regularly over the past 20 years. Immunosuppression related to HIV infection, immunosuppressive treatment, organ transplantation, and neoplastic diseases increases the risk for Leishmania-infected people to develop visceral illness. CASE PRESENTATION: Three cases of Leishmania infantum leishmaniasis in corticosteroid (CS)-treated patients are reported: an isolated lingual leishmaniasis in a farmer treated with CS for asthma, a severe visceral leishmaniasis associated with cutaneous lesions in a woman with myasthenia gravis, and a visceral involvement after cutaneous leishmaniasis in a man receiving CS. CONCLUSION: Physicians should recognise CS-treated patients as a population likely to be immune-suppressed. In immunodeficiency conditions, unusual forms of leishmaniasis can develop and foster the risk of a diagnostic delay and of poor response to therapy.

[Molecular epidemiology of imported malaria in Italy: the use of genetic markers and in vitro sensitivity test in a study of chloroquine resistance in Plasmodium falciparum]. / Epidemiologia molecolare della malaria d'importazione in Italia: l'impiego di marcatori genetici e di saggi di sensibilità in vitro nello studio della clorochino-resistenza in Plasmodium falciparum.

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.

Short Communication: Yersinia pseudotuberculosis septicemia in an HIV-infected patient failed HAART.

The first case of septicemia due to Yersinia pseudotuberculosis in an HIV-infected person was reported. The 42-year-old woman was severely immunosuppressed despite a prolonged exposure to HAART. Specific amplicons for inv, yadA, and lcrF genes showed the pathogenetic potential of the Y. pseudotuberculosis serotype O1 isolate. A favorable clinical response to ceftriaxone and levofloxacin was observed.

An improved PCR-based method for detection and differentiation of Entamoeba histolytica and Entamoeba dispar in formalin-fixed stools.

Detection of infectious agents in faecal samples by polymerase chain reaction (PCR) can be limited by the presence of substances which inhibit DNA amplification. Here, an improved protocol is reported for directly isolating DNA from fresh and aged formalin-fixed stools, after concentration by formalin-ethyl acetate (FEA). The protocol was successfully applied to detect DNA of Entamoeba histolytica/Entamoeba dispar complex in stools by nested PCR, showing high specificity and low detection limit. Extended time of specimen storage in formalin had no influence on PCR yields. This PCR-based method offers technical advantages for routine detection and discrimination of invasive E. histolytica and non-invasive E. dispar.

Strengthening tuberculosis diagnosis in a low-resource setting: experience learned in Dodoma, Tanzania.

INTRODUCTION: Diagnosing tuberculosis in low-resource settings mostly relies on sputum smear microscopy. Improvement through capacity building is a priority. This project aimed to strengthen tuberculosis diagnosis at an intermediate level laboratory. METHODOLOGY: The Italian National Institute for Infectious Diseases and the Italian Development Cooperation closely collaborated with regional and national institutions and reference laboratories to provide laboratory setup, equipment and reagents, personnel training, and the implementation of culture and quality assessment programs at Dodoma Regional Hospital, Dodoma, Tanzania. RESULTS: Microscopy sensitivity was increased, personnel were trained, and culture techniques and quality assessment programs were introduced. CONCLUSIONS: Implementing tuberculosis diagnosis in resource-constrained settings is feasible and represents a basis for further strengthening.