Bridging epidemiology with population genetics in a low incidence MSM-driven HIV-1 subtype B epidemic in Central Europe.

BACKGROUND: The HIV-1 epidemic in Slovenia, a small Central European country, has some characteristics that make it an ideal model to study HIV-1 transmission. The epidemic is predominantly affecting men who have sex with men infected with subtype B (89% of all patients), has a low prevalence (less than 1/1000) and is growing slowly. The aim of the present study was to analyze in detail the evolutionary history and the determinants of transmission. METHODS: A total of 223 partial pol gene sequences from therapy naïve individuals were included, representing 52% of all patients newly diagnosed in 13 years (2000-2012) and analyzed together with genetically similar worldwide sequences, selected in a BLAST search. RESULTS: Combined analysis (maximum likelihood and Bayesian) of HIV-1 transmission chains revealed 8 major clusters (n ≥ 10 patients), 1 group of 4 patients, 2 trios and 12 transmission pairs, thus leaving only 43 (19.3%) Slovenian patients infected with subtype B without a local epidemiological link, indicating a predominance of local transmission of HIV-1 infection. Bayesian analysis performed on a full set of sequences estimated several introductions of HIV-1 into Slovenia, with the most recent common ancestor (tMRCA) of the earliest Slovenian cluster dated to the late 1980s, although tMRCAs obtained from separate independent analysis of each cluster showed considerably more recent estimates. These findings indicate inconsistencies in molecular clock estimation, which we further explored. We hypothesize that these inconsistent dating estimates across the tree could be caused by an evolutionary rate acceleration of HIV-1 after entering the Slovenia epidemic that is not taken into account by the molecular clock model. It could be caused by a lower transmission rate in this setting, as demonstrated by the low epidemic growth rate estimated by Bayesian skyline plot analysis. CONCLUSIONS: HIV-1 subtype B was introduced into Slovenia at several time points from the late 80s onward. The majority of patients had a local transmission link, indicating a closed HIV community. The observed slower epidemic rate suggests that individuals with a long-lasting infection are the driving force of the epidemic in this region.

Severe human granulocytic anaplasmosis transmitted by blood transfusion.

A 36-year-old woman acquired severe human granulocytic anaplasmosis after blood transfusion following a cesarean section. Although intensive treatment with mechanical ventilation was needed, the patient had an excellent recovery. Disease caused by Anaplasma phagocytophilum infection was confirmed in 1 blood donor and in the transfusion recipient.

Azithromycin for acute Q fever in pregnancy.

Q fever is a zoonosis caused by Coxiella burnetii. Although data on Q fever during pregnancy are limited, they indicate that infection with C. burnetii is associated with high morbidity and mortality. The infection is usually asymptomatic in pregnant women but may result in obstetric complications such as spontaneous abortion, intrauterine growth retardation, intrauterine fetal death and premature delivery; in addition, pregnant women are at higher risk of developing chronic Q fever. Treatment of Q fever during pregnancy is challenging not only because C. burnetii is an intracellular bacterium but also because of safety restrictions and limited information on the efficacy of treatment. We report a case of acute Q fever in pregnancy with a successful outcome for mother and child, describe our therapeutic approach to the management of this case, and suggest that treatment with azithromycin may have prevented possible obstetric complications and evolution toward a chronic serologic profile in our patient.

Low prevalence of hepatitis B and C infections among HIV-infected individuals in Slovenia: a nation-wide study, 1986-2008.

BACKGROUND: With the increased lifespan of HIV-1 infected patients, mostly due to highly active antiretroviral therapy, hepatitis C virus (HCV) and hepatitis B virus (HBV) have recently emerged as important pathogens in these patients. HIV-1 infection has an important negative impact on the natural history of HCV and HBV infections, which has consequently caused increased liverassociated and overall morbidity and mortality in HIV-1 infected patients. Thus, liver disease is currently the second leading cause of death in HIV-infected persons in Europe. OBJECTIVE: To determine the prevalence of HBV and HCV infection in HIV-infected individuals in Slovenia. METHODS AND RESULTS: 356 out of 409 Slovenian individuals, confirmed as HIV positive by the end of 2008, were tested for the presence of HBV and HCV infection. Evidence of prior and current HBV infection was found in 77 (21.6%) and 14 (3.9%) of HIV-positive patients, respectively. 38 of 356 (10.7%) HIV-infected individuals were confirmed as anti-HCV positive, and 26 of them (68.4%) were also HCV RNA positive. Concomitant active HBV and HCV infection was found in only two HIV-positive individuals. CONCLUSION: In a study carried out on the highest proportion per entire population of HIV-infected individuals from a certain country or geographical region, Slovenia was identified as the country with the lowest prevalence of HCV infection among HIV-infected individuals.

Severe Clostridium difficile-associated disease in children.

Three cases of Clostridium difficile-associated disease in children were detected within a short time interval. Intensive therapy was required in 2 cases with colectomy in one of them. One of the severe cases was community-acquired. Two patients had underlying diseases (Hirschprung disease, Down syndrome) and also tested positive for enteric viruses (rotavirus, calicivirus).

Effect of rosiglitazone and metformin on insulin resistance in patients infected with human immunodeficiency virus receiving highly active antiretroviral therapy containing protease inhibitor: randomized prospective controlled clinical trial.

AIM: To evaluate and compare effects of 48-week treatment with rosiglitazone and metformin on insulin resistance in patients infected with Human Immunodeficiency Virus (HIV) receiving highly active antiretroviral therapy (HAART), containing a protease inhibitor. METHODS: Randomized prospective controlled clinical trial enrolled 90 male patients infected with HIV and having impaired glucose tolerance and insulin resistance (fasting insulin concentration >20 mIU/L). The patients were randomly assigned into three groups; the first group receiving 4 mg rosiglitazone once a day, the second group receiving 500 mg metformin two times a day, and the third group serving as control without hypoglycemic treatment. The primary efficacy parameters were fasting plasma glucose and insulin levels compared between baseline and week. Data on insulin resistance and beta cell function were analyzed by the Homeostasis Model Assessment (HOMA). RESULTS: After 48 weeks of treatment, the fasting insulin concentration (+/-standard deviation) in rosiglitazone group significantly declined from 39.0+/-3.35 to 19.7+/-3.99 mIU/L (P<0.001; 49% decrease) and in metformin group from 40.3+/-2.29 to 29.2+/-2.82 mIU/L (P<0.001; 27% decrease). HOMA indicated that rosiglitazone significantly reduced insulin resistance from 11.3+/-1.03 to 4.0+/-0.95 (P<0.001), compared with metformin which reduced it from 11.9+/-0.73 to 5.7+/-0.62 (P<0.001). Insulin resistance was significantly lower in the rosiglitazone group after 48 weeks (P<0.001). Metformin significantly improved beta cell function (from 257.3+/-21.91 to 707.4+/-207.32; P<0.001), as did rosiglitazone as well (from 261.3+/-27.98 to 403.3+/-162.50; P<0.001), but the improvement in the metformin group was significantly better (P<0.001). However, metformin was more efficient in improving beta cell function (from 257.3+/-21.91 to 707.4+/-207.32) than rosiglitazone (from 261.3+/-27.98 to 403.3+/-162.50). CONCLUSIONS: Both rosiglitazone and metformin were effective and well tolerated in HIV treated with protease inhibitor-containing HAART. Rosiglitazone significantly more reduced insulin resistance, while beta cell function was significantly better in patients on metformin. Both drugs may be considered as an appropriate therapy, with rosiglitazone being a better alternative in treating insulin resistance in this patient population. ClinicalTrials.gov trial registration number: NCT00483392.

Prevalence of antiretroviral drug resistance mutations and HIV-1 non-B subtypes in newly diagnosed drug-naïve patients in Slovenia, 2000-2004.

In order to estimate the prevalence and patterns of antiretroviral drug resistance mutations in drug-naïve HIV-1 infected patients in Slovenia, and the prevalence of non-B subtypes, a retrospective study was conducted on a cohort, representing 87% of the total of newly diagnosed HIV-1 infected patients, in a 5 year period (2000-2004). Protease (PR) and reverse transcriptase (RT) sequences were determined in 77 newly diagnosed HIV-1 patients. Non-B subtypes were present in 18% of the population tested. Transmitted drug resistance was identified as in the CATCH study: the presence of primary PR and RT gene mutations according to the IAS-USA mutation list including the revertant mutations in codon 215 and excluding mutations on the RT positions 44 and 118. The estimated prevalence of transmitted resistance mutations was 3.9%. Namely, three out of 77 patients had mutations associated with resistance to NRTIs: one patient carried M184V in association with A62V, while a revertant mutation T215D was found in two patients. No transmitted drug resistance to NNRTIs or PIs was detected. However, to score the expected response to therapy using the REGA and the Stanford algorithms, we also took into account secondary PR mutations and additional RT mutations. Reduced response to some therapeutic options was predicted in five patients (6.5%). In conclusion, testing the vast majority of all newly diagnosed HIV-1 patients in the last 5 years in Slovenia uncovered a relatively high prevalence of non-B subtypes and a low prevalence of transmitted drug resistance.

Severe tick-borne encephalitis in Slovenia in the years 2001-2005: time for a mass vaccination campaign?

The aim of this retrospective study was to assess some clinical, epidemiological and laboratory parameters of severe tick-borne encephalitis in Slovenia in the last five years, to compare them with published data, and to estimate need for providing a policy of active immunization. Thirty-three adult patients with a severe course of the disease, admitted to the intensive care unit of the Department of Infectious Diseases, University Medical Centre Ljubljana, Slovenia during a five year period, participated. All the patients had specific serum IgM antibodies against tick-borne encephalitis virus at admissions and IgG antibodies were present in 29 out of 33 patients. Twenty-two patients were admitted because of severe consciousness disturbances, nine suffered from spinal nerve paralysis, in two patients cranial nerve paralysis was observed, and one suffered from generalized tonic-clonic seizures. Ten patients were mechanically ventilated and three died. Leukocytosis in peripheral veins was found in twenty-one patients and nine had a C-reactive protein serum concentration over 50 mg/l. Nineteen patients had a cerebrospinal fluid leukocyte count exceeding 100 x 10(6)/l and a cerebrospinal fluid protein concentration was over the cut-off value of 0.45 g/l in majority. The findings of the present study confirmed some previous reports about clinical, epidemiological and laboratory characteristics of patients with severe tick-borne encephalitis. We have found that tick-borne encephalitis in Slovenia has a relatively low fatality rate. However, the severe course with long-lasting sequelae of the disease justifies vaccination of a risk population in endemic areas.

Effect of metformin and rosiglitazone on lipid metabolism in HIV infected patients receiving protease inhibitor containing HAART.

AIMS: Insulin resistance may be the primary event in the protease inhibitor-associated metabolic syndrome. Treatment with insulin sensitizers (metformin, rosiglitazone) can ameliorate insulin resistance. So far, the effects of these agents on blood lipids have not been well determined. The aim of the present study was to evaluate the effects of metformin and rosiglitazone treatment on lipid metabolism in HIV infected patients receiving protease inhibitors containing HAART. DESIGN AND METHODS: HIV infected male patients (>18 years) were eligible for the study if they had impaired glucose tolerance with insulin resistance, characterized by fasting insulin concentration greater then 20 mIU/L and if they were on stable antiretroviral therapy regimen including a protease inhibitor for at least 12 months prior to the study enrolment. The patients were randomly assigned to receive either 1g/day metformin (metformin group, n=30) or 4 mg/day rosiglitazone maleate (rosiglitazone group, n=30) or no treatment (control group, n=30). The primary efficacy parameters were fasting plasma lipids, glucose levels and fasting insulin levels compared between baseline and week 48, by treatment groups. RESULTS: The total cholesterol concentration in rosiglitazone group increased from 5.76 -/+1.2 to 7.1-/+1.6 mmol/l (23% increase, p<0.05), HDL levels increased from 0.91-/+0.44 to 1.3-/+0.2 (38% increase, p<0.01) and LDL levels increased from 3.5-/+0.98 to 4.5-/+1.0 (28% increase, p<0.05). Treatment with metformin had no significant effect on total, HDL and LDL cholesterol. After 48 weeks of treatment, the fasting triglycerides concentration in the metformin group declined from 4.1-/+1.6 to 3.2-/+1.3 mmol/l (22% decrease,p<0.05) but in the rosiglitazone group no statistically significant effect on plasma triglycerides was noted. Furthermore, after 48 weeks of treatment the fasting insulin concentration in the rosiglitazone group declined by 49% and in the metformin group by 28%. This improvement in insulin secretion could be clearly demonstrated when the sums of insulin concentrations after oral glucose tolerance test were compared: 548-/+13 to 345-/+11.8 mIU/l in the rosiglitazone group (37% decrease, p<0.01) and from 552-/+15 to 420-/+12 mIU/l in the metformin group (24% decrease, p<0.01). CONCLUSIONS: The study demonstrates that both therapies improve insulin resistance. However, treatment with metformin has no effect on total, HDL and LDL cholesterol, but significantly reduces triglycerides, which has beneficial effect on the lipid status in these patients. Rosiglitazone causes significant increases in total cholesterol, HDL and LDL, but has no effect on triglycerides concentrations.

Severe tick-borne encephalitis in Slovenia: epidemiological, clinical and laboratory findings.

The aim of this retrospective study was to determine epidemiological, clinical and laboratory characteristics in adult patients with the severe form of tick-borne encephalitis. Thirty-one patients with a severe course of disease admitted to the intensive care unit of the Department of Infectious Diseases at the University Medical Centre Ljubljana, Slovenia, between 1996 and 2000 were included in the study. Tick-borne encephalitis virus infection was confirmed by the demonstration of specific IgM and IgG antibodies in serum using routine serological screening tests. All the patients had specific IgM antibodies at admission and IgG antibodies were present in 90.3%. Nineteen patients suffered from severe meningoencephalitis, 11 from meningoencephalomyelitis, and dysfunction of the autonomic nervous system was found in one patient. The mortality rate was 3.3%. Tick bite was recorded in 58% and a characteristic biphasic course of the disease was found in 16 patients. The median cerebrospinal fluid leukocyte count was 112 x 10(6)/l, and in 93.5% of patients the cerebrospinal fluid protein concentration was greater than the cut-off value of 0.45 g/l. The findings of the present study indicate some clinical, epidemiological and laboratory distinctions between published data on the mild or moderately severe form of tick-borne encephalitis and our patients with severe disease.

Lipodystrophy and metabolic abnormalities in Slovenian HIV-infected patients.

The purpose of this cross-sectional survey was to estimate the prevalence of lipodystrophy (LD) and metabolic abnormalities in Slovenian patients with HIV infection. All patients receiving highly active antiretroviral therapy (HAART) for more than six months (treated group) and all known antiretroviral-naive patients (control group) were consecutively evaluated between October and December 2003. Eighty-one treated patients (81% male, 19% female), and 18 controls (83% male, 17% female) were included in the study. In the treated group, the duration of HAART at the time of evaluation was 3.7 +/- 2.3 years. Twenty-nine treated patients (36%) had at least one sign of LD: isolated peripheral atrophy was present in nine (31%), isolated fat accumulation in four (14%) and a mixed syndrome in 16 (55%). Patients with evidence of LD were older than those without LD and had a higher prevalence of AIDS and a longer duration of HAART, but there were no differences in HIV transmission categories, plasma RNA level, CD4+ count, HAART regimens or BMI. Insulin resistance was observed in 31 treated patients (38%), of whom 22 (27%) had impaired glucose tolerance and six (7%) had diabetes mellitus. Dyslipidemia was the predominant metabolic abnormality in the treated group, observed in 58 patients (72%). Levels of total cholesterol were increased in 43 patients (53%), and hypertriglyceridemia was noted in 40 (49%). The duration of HAART in patients with metabolic syndrome was longer than in patients without the syndrome. Lipid- and glucose-related abnormalities were more frequent in patients with LD than in those without. A total of 60 treated patients (74%) had at least one sign of LD and/ or one metabolic alteration at the time of evaluation. In the control group, none of the patients showed evidence of LD, and metabolic abnormalities were less common than in the treated group: six patients (33%) had one or more metabolic abnormalities. HIV-related LD syndrome includes a variety of clinical and biological manifestations, which can be included in a case definition. The metabolic effects of HAART could lead to an increase in cardiovascular disease. The patient's metabolic parameters should be evaluated before starting treatment, and appropriate management of LD and glucose- or lipid-related metabolic changes is essential.

Cardiovascular risk assessment in HIV-infected male patients: a comparison of Framingham, SCORE, PROCAM and DAD risk equations.

INTRODUCTION: Traditional cardiovascular (CVD) risk assessment algorithms such as the Framingham Risk Score (FRS), Systematic Coronary Risk Evaluation (SCORE) and Prospective Cardiovascular Munster (PROCAM) were developed for general populations, their usefulness in HIV-infected population has not been confirmed. DAD algorithm was developed specifically for HIV-infected patients. The aim of our study was to evaluate the performance of risk assessment algorithms in HIV-infected population. METHODS: A prospective cross-sectional national study that included 83 HIV-infected male patients from Slovenia below the age of 55 was performed. CVD risk was assessed using four algorithms, the presence of subclinical atherosclerosis was determined by measuring carotid intima-media thickness (CIMT); patients were followed up for 5 years. RESULTS: High proportion of patients with low CVD risk according to FRS (61.9%) and PROCAM (81.0%) and only 7.1% according to SCORE had evidence of subclinical atherosclerosis. Only 7.1% of patients with low CVD risk according to DAD algorithm had evidence of subclinical atherosclerosis. CONCLUSION: Our study has shown that SCORE and DAD algorithm were superior to FRS and PROCAM. In younger HIV-infected patients, even with moderate CVD risk, CIMT assessment should be employed in a complete clinical evaluation as a more aggressive prevention and treatment approach is warranted.

Relationship between markers of endothelial dysfunction and inflammation and subclinical atherosclerosis in HIV-infected male patients below 55 years of age.

INTRODUCTION: Antiretroviral therapy in HIV-infected patients appears to be associated with increased incidence of cardiovascular disease (CVD).The aim of our study was to investigate the differences in markers of inflammation, endothelial dysfunction and prothrombotic state between treated and untreated HIV-infected patients with or without subclinical atherosclerosis. METHODS: Eighty-six Slovenian HIV-infected male patients below the age of 55 participated in our study. Levels of high-sensitivity C-reactive protein (hsCRP), vascular cell adhesion molecule 1 (VCAM-1) and plasminogen activator inhibitor 1 (PAI-1) were measured. The presence of subclinical atherosclerosis was determined by measuring carotid intima-media thickness. RESULTS: The level of hsCRP was significantly increased in HIV-infected patients; it was higher in treated than untreated patients. VCAM-1 was significantly increased; it was higher in untreated than treated patients. PAI 1 was significantly increased; there were no differences between untreated and treated patients. Patients with subclinical atherosclerosis had elevated hsCRP; levels of VCAM-1 and PAI-1 were not significantly different. CONCLUSION: Signs of systemic and vascular inflammation persist in both untreated and treated HIV infected patients. None of the studied markers contributed to improved assessment of subclinical atherosclerosis. The usefulness of such markers in routine clinical evaluation of CVD risk in HIV infected patients remains unclear.

Early atherosclerosis in HIV-infected patients below the age of 55 years: Slovenian national study.

BACKGROUND: Increased life expectancy of human immunodeficiency virus (HIV)-infected patients appears to be coupled with increased incidence of cardiovascular disease (CVD). AIM: The aim of our study was to determine the presence of early atherosclerosis among Slovenian HIV-infected patients below the age of 55 years. METHODS: A total of 86 HIV-infected male patients below the age of 55 years participated in our study. Ankle-brachial index (ABI) was measured using a handheld Doppler ultrasonic probe and a blood pressure cuff. Carotid intima-media thickness (CIMT) was assessed by the B-mode high-resolution ultrasound technique. Low ABI, CIMT > 0.8 mm or presence of carotid plaques were considered markers of early atherosclerosis. RESULTS: Average CIMT was lowest among treatment-naïve patients (0.65 mm); 10 (38.4 %) had CIMT > 0.8 mm, and carotid plaques were detected in 1 (3.8 %). Average CIMT among treated patients was 0.71 mm; 30 (50.0 %) had CIMT > 0.8 mm, and plaques were detected in 11 (18.3 %). Low ABI (≤ 0.90) was found in five patients (5.8 %) without symptoms of peripheral artery disease; two were treatment-naïve, and three received antiretroviral therapy. Early atherosclerosis was found in 43 (50.0 %) patients; 10 (38.4 %) were in treatment-naïve and 33 (55.0 %) in the treated group. CONCLUSIONS: Increased prevalence of early atherosclerosis among Slovenian HIV-infected patients below the age of 55 years has been demonstrated. Screening for early atherosclerosis should be implemented in the evaluation of young HIV-infected patients because a more aggressive treatment approach, aimed to delay the progression of atherosclerosis, may be warranted especially when carotid plaques are detected. We have shown that although ABI contributes to CVD risk assessment, CIMT assessment remains the more sensitive method.

Short communication: prevalence of HIV type 1 transmitted drug resistance in Slovenia: 2005-2010.

Slovenia is a small European country with a total of 547 HIV-infected individuals cumulatively reported by the end of 2011. However, the estimated incidence rate of HIV infections increased from 7.0 per million in 2003 to 26.8 per million in 2011. In this study, we assessed the prevalence of transmitted drug resistance (TDR) in the past 6 years (2005-2010) and analyzed the time trend of the proportion of men having sex with men (MSM) and HIV-1 subtype B among newly diagnosed individuals in a 15-year period (1996-2010) in Slovenia. Among 150 patients included in the study, representing 63% of HIV-1 newly diagnosed patients in 2005-2010, TDR was found in seven patients (4.7%). The prevalence of TDR to nucleoside reverse transcriptase inhibitors, nonnucleoside reverse transcriptase inhibitors, and protease inhibitors was 2% (3/150), 2% (3/150), and 0.7% (1/150), respectively. The majority of patients were infected with subtype B (134/150, 89%), while subtype A was detected in 6.0% (9/150), subtype D in 1.3% (2/150), and subtype G and CRF02_AG in 0.7% (one patient each). Three of 150 sequences could not be typed. Infection with subtype B was found to be significantly associated with male gender, Slovenia being reported as the country of the patient's nationality and origin of the virus, CDC class A, mode of transmission with homosexual/bisexual contact, sex with an anonymous person, and a higher CD4(+) count. Among patients carrying the subtype B virus, an MSM transmission route was reported in 87% of patients. Although the prevalence of TDR in Slovenia is still below the European average, active surveillance should be continued, especially among MSM, the most vulnerable population for HIV-1 infection in this part of Europe.

Lactococcus garvieae septicaemia in a patient with artificial heart valves.

Lactococcus garvieae is usually an animal pathogen. Only a few cases of infections in humans have been described. We describe a case of an elderly patient with prosthetic heart valves with a septicaemia without infective endocarditis, and with a favourable clinical course.

Prevalence and risk factors for osteopenia/osteoporosis in an HIV-infected male population.

The objective of our investigation was to estimate the prevalence of osteopenia/osteoporosis in men with HIV/AIDS and evaluate the role of antiretroviral treatment (ART), HIV and other risk factors in reducing bone mineral density (BMD). All known Slovenian HIV-infected ART-naïve and treated males (infected or treated > 12 months) were invited to participate in a cross-sectional study. Data were collected on age, BMI, waist-hip ratio, family history of hip fracture, duration of infection, duration of ART, smoking, alcohol, exercise, viral load and CD4+ cells. BMD was measured using dual X-ray absorptiometry. A total of 96 patients (out of 133 who fulfilled the inclusion criteria) were assessed and allocated into three groups: group A (n = 24), ART-naïve; group B1 (n = 37), treated with non-protease-inhibitor (PI) containing ART; and group B2 (n = 35), treated with PI-containing ART. The prevalence of osteopenia/osteoporosis was 57/96 (59%): osteopenia 45/96 (47%) and osteoporosis 12/96 (12%). Significantly lower BMD was detected in group A (P = 0.020). Multiple logistic regression analysis showed ART to be an independent negative predictor for reduced BMD (P = 0.037; OR = 0.29, 95%CI 0.09-0.93). Vitamin D(3) deficiency was detected in 79 (82%) of the patients. The study group represented 72% of the national HIV-infected male population; this proportion being higher than in any other study reported to date. The prevalence of reduced BMD was notably higher than the national prevalence among men of comparable age. There was no association between reduced BMD and any specific ART. According to our results, absence of ART was confirmed as an independent predictor of osteopenia/osteoporosis. Targeted screening and early treatment present a reasonable strategy for preventing reduced BMD in HIV-infected patients, but correcting vitamin D(3) levels could also be an important component.