Course of Plasmodium infection studied using 2D-COS on human erythrocytes.

BACKGROUND: The threat of malaria is still present in the world. Recognizing the type of parasite is important in determining a treatment plan. The golden routine involves microscopic diagnostics of Giemsa-stained thin blood smears, however, alternative methods are also constantly being sought, in order to gain an additional insight into the course of the disease. Spectroscopic methods, e.g., Raman spectroscopy, are becoming increasingly popular, due to the non-destructive nature of these techniques. METHODS: The study included patients hospitalized for malaria caused by Plasmodium falciparum or Plasmodium vivax, in the Department of Infectious Diseases at the University Hospital in Krakow, Poland, as well as healthy volunteers. The aim of this study was to assess the possibility of using Raman spectroscopy and 2D correlation (2D-COS) spectroscopy in understanding the structural changes in erythrocytes depending on the type of attacking parasite. EPR spectroscopy and two-trace two-dimensional (2T2D) correlation was also used to examine the specificity of paramagnetic centres found in the infected human blood. RESULTS: Two-dimensional (2D) correlation spectroscopy facilitates the identification of the hidden relationship, allowing for the discrimination of Raman spectra obtained during the course of disease in human red blood cells, infected by P. falciparum or P. vivax. Synchronous cross-peaks indicate the processes taking place inside the erythrocyte during the export of the parasite protein towards the cell membrane. In contrast, moieties that generate asynchronous 2D cross-peaks are characteristic of the respective ligand-receptor domains. These changes observed during the course of the infection, have different dynamics for P. falciparum and P. vivax, as indicated by the asynchronous correlation cross-peaks. Two-trace two-dimensional (2T2D) spectroscopy, applied to EPR spectra of blood at the beginning of the infection, showed differences between P. falciparum and P. vivax. CONCLUSIONS: A unique feature of 2D-COS is the ability to discriminate the collected Raman and EPR spectra. The changes observed during the course of a malaria infection have different dynamics for P. falciparum and P. vivax, indicated by the reverse sequence of events. For each type of parasite, a specific recycling process for iron was observed in the infected blood.

Bacterial meningitis in adults - principles of diagnosis and antibiotic treatment.

The emergence of antibiotic-resistant bacterial strains in recent years has necessitated the development of new strategies for empiric antimicrobial therapy for bacterial meningitis. Bacterial meningitis is associated with significant morbidity and mortality despite the availability of effective antimicrobial therapy. The management approach to patients with suspected or proven bacterial meningitis includes initiation of appropriate antimicrobial and adjunctive therapies and decides on the patients survival.

Real-world effectiveness and safety of direct-acting antivirals in patients with cirrhosis and history of hepatic decompensation: Epi-Ter2 Study.

BACKGROUND AND AIMS: The aim of this study was to assess the real-life effectiveness and safety of direct acting antivirals (DAAs) in patients with cirrhosis and history of hepatic decompensation compared to those with compensated cirrhosis. METHOD: Data of patients treated with DAAs and included in the EpiTer-2 database (N = 10 152) were collected retrospectively. The primary endpoint was sustained viral response (SVR) at 12 weeks posttreatment. Patients were also evaluated in terms of liver-related adverse events and treatment modification/discontinuation. RESULTS: The overall SVR rate was 91.4% in the intent to treat (ITT) analysis and 95.2% in the per-protocol (PP) analysis (P < .001). Patients with decompensated cirrhosis had lower SVR rates compared to those with compensated cirrhosis in ITT analysis (86.4% vs 92.0%, P < .001), while not in PP analysis (92.9% vs 95.5%, P > .05). Adverse events (AE) occurred 45.6% and 29.3% of patients with decompensated and compensated cirrhosis (P < .001). Patients with decompensated cirrhosis were at higher risk of death (5.4% vs 0.9%; P < .0001) or liver decompensation (21.5% vs 1.3%; P < .0001). Treatment with protease inhibitors was not associated with hepatic decompensation (P = .3). Only 82.6% of patients with decompensated cirrhosis completed DAA treatment (vs 92.8% in compensated cirrhotics; P < .0001). CONCLUSION: Despite higher frequency of AE and treatment modifications, once completed, DAAs yield comparable results for patients with decompensated and compensated cirrhosis. High rate of serious adverse events in patients with advanced liver disease treated with PI may not be related to the detrimental effect of the medications, but rather to the disease itself.

Neurological symptoms in hospitalised patients with COVID-19 and their association with in-hospital mortality.

OBJECTIVES: To evaluate the spectrum of neurological symptoms in patients with COVID-19 during the first 14 days of hospitalisation and its association with in-hospital mortality. MATERIAL AND METHODS: We included 200 patients with RT-PCR-confirmed COVID-19 admitted to University Hospital in Krakow, Poland. In 164 patients, a detailed questionnaire concerning neurological symptoms and signs was performed prospectively within 14 days of hospitalisation. In the remaining 36 patients, such questionnaires were completed retrospectively based on daily observations in the Department of Neurology. RESULTS: During hospitalisation, 169 patients (84.5%) experienced neurological symptoms; the most common were: fatigue (62.5%), decreased mood (45.5%), myalgia (43.5%), and muscle weakness (42.5%). Patients who died during hospitalisation compared to the remainder were older (79 [70.5-88.5] vs. 63.5 [51-77] years, p = 0.001), and more often had decreased level of consciousness (50.0% vs. 9.3%, p < 0.001), delirium (33.3% vs. 4.4%, p < 0.001), arterial hypotension (50.0% vs. 19.6%, p = 0.005) or stroke during (18.8% vs. 3.3%, p = 0.026) or before hospitalisation (50.0% vs. 7.1, p < 0.001), whereas those who survived more often suffered from headache (42.1% vs. 0%, p = 0.012) or decreased mood (51.7% vs. 0%, p = 0.003). CONCLUSIONS: Most hospitalised patients with COVID-19 experience neurological symptoms. Decreased level of consciousness, delirium, arterial hypotension, and stroke during or before hospitalisation increase the risk of in-hospital mortality.

Real-world experience with Grazoprevir/Elbasvir in the treatment of previously "difficult to treat" patients infected with hepatitis C virus genotype 1 and 4.

BACKGROUND AND AIM: Grazoprevir/elbasvir (GZR/EBR) was approved for the treatment of chronic hepatitis C virus (HCV) genotype 1 and 4 infected patients with or without compensated liver cirrhosis. The aim of this study was to assess GZR/EBR regimen in the real-world experience, particularly in previously "difficult-to-treat" patients with chronic kidney diseases, human immunodeficiency virus-coinfected, cirrhotics, and treatment-experienced. METHODS: The analysis included patients treated with GZR/EBR selected from 10 152 individuals from the EpiTer-2 database, large national real-world study evaluating antiviral treatment in 22 Polish hepatology centers between 2015 and 2018. Data were completed retrospectively and submitted online. RESULTS: A total of 1615 patients who started GZR/EBR therapy in 2017 and 2018 with a female predominance (54%) and median age of 54 years were analyzed. The majority were infected with GT1b (89%) and treatment naïve (81%). Liver cirrhosis was diagnosed in 19%, and 70% of patients had comorbidities, of which chronic renal disease was present in 7% and HIV-coinfection in 4%. Overall, a sustained virologic response (SVR) was achieved by 95% according to intent-to-treat (ITT) and 98% after exclusion of lost to follow up (modified ITT). No differences were found in cure rate between all included patients and subpopulations previously considered as difficult-to-treat. Majority of patients completed the treatment course as scheduled, adverse events were mostly mild and did not lead to therapy discontinuation. CONCLUSIONS: GZR/EBR treatment carried-out in patients infected with HCV genotype 1 and 4 demonstrated good tolerability and an excellent SVR rate with no effectiveness reduction in so called difficult-to-treat populations.

Management of bacterial skin and soft tissue infections.

Skin and soft tissue infections (SSTIs) are a group of diseases usually caused by bacteria, and connected with different clinical picture, course, and prognosis. The increasing incidence of SSTIs is associated mainly with aging of the population, the increasing number of metabolic diseases, especially diabetes mellitus, as well as cardiovascular diseases. Although SSTIs are often benign and usually does not require medical consultations, some of them may cause a systemic infection. In this situation, knowledge of the principles of diagnostic work-up and therapy is essential. The principles of recognition and treatment of skin and soft tissue infections, including new biocidal drugs, are presented.

Clostridium difficile infection: review.

Clostridium difficile (C. difficile) is a Gram-positive, spore-forming, anaerobic bacillus, which is widely distributed in the intestinal tract of humans and animals and in the environment. In the last decade, the frequency and severity of C. difficile infection has been increasing worldwide to become one of the most common hospital-acquired infections. Transmission of this pathogen occurs by the fecal-oral route and the most important risk factors include antibiotic therapy, old age, and hospital or nursing home stay. The clinical picture is diverse and ranges from asymptomatic carrier status, through various degrees of diarrhea, to the most severe, life threatening colitis resulting with death. Diagnosis is based on direct detection of C. difficile toxins in feces, most commonly with the use of EIA assay, but no single test is suitable as a stand-alone test confirming CDI. Antibiotics of choice are vancomycin, fidaxomicin, and metronidazole, though metronidazole is considered as inferior. The goal of this review is to update physicians on current scientific knowledge of C. difficile infection, focusing also on fecal microbiota transplantation which is a promising therapy.

Fatty acids and selected endocannabinoids content in cerebrospinal fluids from patients with neuroinfections.

Neuroinfections are a significant medical problem and can have serious health consequences for patients. Their outcome, if not fatal, can be associated with permanent residual deficits. Cerebrospinal fluid (CSF) examination is commonly used for meningitis confirmation. Fatty acids (FA) are precursors of lipid mediators with pharmacological activity. They actively modulate inflammation as well as contribute to its resolution. Therefore the aim of this study was to determine the FA and selected endocannabinoids (ECB) content in the CSF obtained from patients with bacterial (BM) and viral meningitis (VM) using chromatographic techniques. A significantly lower level of saturated FA was found in patients with BM and VM as compared to controls. There was a significantly higher concentration of long-chain monounsaturated FA and polyunsaturated n-6 FA in the CSF obtained from patients with neuroinfection. Moreover, a significant reduction of n-3 FA in CSF obtained from patients with BM and VM was demonstrated. The highest amount of ECB was detected in the CSF of patients with VM: eicosapentaenoyl ethanolamide (1.65 pg/mL), docosahexaenoyl ethanolamide (655.5 pg/mL) and nervonoyl ethanolamide (3.09 ng/mL). Results indicate the participation of long-chain monounsaturated and polyunsaturated FA and their derivatives in the inflammatory process and likely in the process of resolution of inflammation during neuroinfection. It seems that the determination of the FA and ECB profile in CSF may be a valuable biomarker of health and may allow the development of new pharmacological strategies, therapeutic goals and fatty acids supplementation necessary in the fight against inflammation of the central nervous system.

Meningoencephalitis caused by Lactobacillus plantarum - case report.

Specific strains of Lactobacillus spp. are widely used as probiotic agents but it has been repeatedly reported that may have a pathogenic potential. We present the report on a case of meningoencephalitis caused by Lactobacillus plantarum in a 63-year-old man with newly diagnosed metastatic planoepitheliale lung cancer. The patient was hospitalised due to newly diagnosed cancer and during the course of hospitalisation developed symptoms of neuroinfection. On the basis of the symptoms and results of the conducted tests the patient was diagnosed with bacterial meningoencephalitis. In microbiological tests of the blood and cerebrospinal fluid L. plantarum was cultured. During the course of antibiotic therapy the patient's condition improved. Lactobacilli are now recognised as a causative agent of infection, most notably bacteraemia. To our knowledge, this is the fourth documented case of Lactobacillus-associated neuroinfection, and only the second in an adult. Lactobacilli cause mostly opportunistic infections in immunocompromised individuals.

Differences between palpation and static/dynamic tests to diagnose painful temporomandibular disorders in patients with Lyme disease.

OBJECTIVES: The aim was to determine the frequency of Research Diagnostic Criteria for Temporomandibular Disorders (RDC/TMD)-based pain diagnoses and dynamic/static tests-based pain diagnoses, and to assess the agreement of palpation tests with static/dynamic tests. MATERIALS AND METHODS: Eighty-six (N = 86) adult patients with Lyme disease (mean age 57.0 ± 14.3 years; male/female ratio was 42/44) were examined according to techniques described in the RDC/TMD. Additionally, dynamic/static tests were performed. For RDC/TMD-based pain diagnoses and dynamic/static tests-based pain diagnoses, descriptive frequencies were calculated. Differences between the frequency of palpation-based diagnoses and of dynamic/static-based diagnoses as well as the agreement between pain diagnoses established with the two diagnostic approaches were assessed. RESULTS: RDC/TMD-based pain diagnoses were made in 61 patients for myofascial pain and in 11 patients for arthralgia and/or osteoarthritis. Based on dynamic/static tests, mainly myogenous pain was diagnosed in 6 patients, and a mainly arthrogenous pain in 5. The agreement of palpation tests with static/dynamic tests in Lyme disease population was poor. CONCLUSION: A high prevalence of TMD symptoms was found in patients with Lyme disease. The results suggest that using palpation tests alone could overestimate primary TMDs when comorbid conditions are present. CLINICAL RELEVANCE: Dynamic/static tests should be used as part of the routine TMD assessment. In case of Lyme disease as the actual cause of the facial pain, while the dentist might be suspecting TMD when dynamic/static TMD tests are negative, referral to an appropriate specialist for the diagnosis and treatment of Lyme disease needs to be made.

Analysis of cases of Lyme arthritis in patients hospitalized in Infectious Diseases Department, University Hospital in Cracow.

Lyme disease is an emerging problem in Poland. Analysis has been undertaken of the medical documentation of 86 patients hospitalized in the Infectious Diseases Department, University Hospital in Cracow in 2013-2016, suspected of Lyme arthritis. It has especially considered medical history including potential exposure to the infection, detailed characteristics of the symptoms, diagnostic challenges and results of the treatment. Only some patients had a history of erythema migrans and not all of them recalled tick-bite. The majority of the patients had affected large joints, especially knee joints, and polyarthritis was rarely observed. Symptoms were resolved completely or partially after antibiotic treatment in most patients. The diagnosis of Lyme arthritis in areas endemic for Lyme disease is still a diagnostic challenge in patients with other rheumatic diseases, including osteoarthritis.

The level of fecal calprotectin significantly correlates with Clostridium difficile infection severity.

INTRODUCTION: Fecal calprotectin (FC) rises significantly in intestinal inflammation accompanied by neutrophil activation - such as Clostridium difficile infection (CDI). The aim of the study was to evaluate the benefit of FC testing in assessing the severity of CDI. MATERIALS AND METHODS: The study group included 76 patients with CDI hospitalized in the Jagiellonian University Hospital in Krakow from July 2017 till January 2018. FC levels were measured using an EIA (Enzyme Immunoassay). Demographic, clinical information and blood tests were recorded using standardized data collection forms. The selection of patients into non-severe and severe groups was carried out in accordance with the ESCMID criteria (European Society of Clinical Microbiology and Infectious Diseases) and some modi cations to those criteria were proposed. RESULTS: the studied population included 76 patients (39 men and 37 women) with CDI aged from 24 to 98 years (mean: 72). Median calprotectin level was 739 (Q25-Q75: 612-799 µg/g), characteristic of patients with colitis. A statistically significant difference in FC concentration in patients with severe vs non-severe CDI was observed (severe - 770 vs non-severe - 659 µg/g, p = 0.009). FC directly correlated with platelets level; however, no correlation between FC level and the blood parameters prognostic for CDI (leukocyte, neutrophil count, albumin, creatinine levels) was found. CONCLUSION: FC level is an indication of ongoing intestinal inflammation in CDI patients. FC level significantly correlated with CDI severity, which demonstrates that FC could serve as a predictive marker for assessing CDI severity.

The presence of IL-8 +781 T/C polymorphism is associated with the parameters of severe Clostridium difficile infection.

PURPOSE: There is large variation in the clinical manifestations of Clostridium difficile infection (CDI). We also still can not predict which patients are more susceptible to reinfection with CDI. The aim of our study was to evaluate the effect of gene single nucleotide polymorphisms (SNP) of proinflammatory cytokines, specifically IL-1ß, IL-8 on the development, clinical course and recurrence of CDI. METHODS: We performed a prospective study of adults (130 people ≥ 18 years) including 65 patients with CDI treated in tertiary hospital and 65 healthy persons. The following 3 variants were analyzed for the occurrence of gene polymorphisms in patients with CDI versus the control group: IL-1ß +3953 A/G (rs1143634), IL-1ß -31 A/G (rs1143627), and IL-8 +781 T/C (rs2227306). Then, we assessed the correlation between these genetic polymorphisms and biochemical parameters important in CDI course, CDI severity as well as CDI recurrence. RESULTS AND CONCLUSIONS: The presence of genetic polymorphisms of IL-1ß +3953 A/G, -31 A/G and IL-8 +781 T/C did not have an effect on the development or recurrence of CDI. The presence of IL-8 +781 T/C polymorphism is associated with the severe CDI.

The efficacy of paritaprevir/ritonavir/ombitasvir+dasabuvir and ledipasvir/sofosbuvir is comparable in patients who failed interferon-based treatment with first generation protease inhibitors - a multicenter cohort study.

BACKGROUND: According to the EASL and AASLD guidelines, the recommended treatment for patients who failed to achieve a sustained virologic response (SVR) on prior interferon-based triple therapy with protease inhibitors (PI), is a combination of sofosbuvir and NS5A inhibitors. Polish national recommendations also allow the use of paritaprevir/ritonavir/ombitasvir+dasasbuvir±ribavirin (PrODR) in this group of patients. The aim of the study was to evaluate the efficacy and safety of PrODR vs. ledipasvir/sofosbuvir±RBV (LSR) in PI-experienced patients in real-life setting. METHODS: Our analysis included patients registered in the nationwide, investigators initiated, multicentre EpiTer-2 database. Among 4530 patients registered, 335 with genotype 1 (93% 1b) were previously treated with IFN-based regimens with PIs: 127 with boceprevir (BOC), 208 with telaprevir (TVR). Patients with advanced fibrosis (F3/F4) were significantly predominant (BOC 28.4%/61.4%, TVR 18.8%/64.4%, respectively). Subjects were assigned to IFN-free retreatment as follows: BOC - 64 (50.4%) PrODR and 63 (49.6%) LSR; TVR- 103 (49.5%) PrODR and 105 (50.5%) LSR. RESULTS: SVR rates were comparable for particular groups: BOC → PrODR- 100%; BOC → LSR - 98%; TVR → PrODR - 97%; TVR → LSR - 96% (intent-to treat analysis-ITT) and BOC → PrODR→100%; BOC → LSR - 99%; TVR → PrODR - 99%; TVR → LSR - 98% (modified intent-to treat analysis-mITT). Both treatment regimens had a favourable safety profile. Adverse events (AEs) were generally mild or moderate in severity. Three deaths were reported. The treatment was stopped due to AEs in five patients (three treated with PrODR and two with LSR). CONCLUSION: Efficacy and safety of treatment with PrODR and LSR is comparable in BOC or TVR-experienced patients.

Analysis of risk factors and outcomes of Clostridium difficile infection.

INTRODUCTION: Clostridium difficile (C. difficile) is a Gram-positive, anaerobic rod-shaped bacteria, widely spread in the human environment. In the last decade, the frequency and severity of Clostridium difficile infection (CDI) have been increasing, making this particular disease one of the most significant nosocomial infections. The aim of our study was an analysis of CDI risk factors, its course and consequences. MATERIALS AND METHODS: Medical documentation of the patients treated for CDI in the University Hospital in Cracow and St Anne's Hospital in Miechów has been analysed. The analysis focused on epidemiological data, blood parameters, comorbidities, recurrence rate, and complication rate (deaths included). As part of risk factors analysis, antibiotic use or hospitalisation in a period of 3 months before the episode of infection was considered relevant. Blood tests have been performed using routinely employed, standard methods. RESULTS: We evaluated data of 168 people infected with C. difficile, out of which there were 102 women (61%) and 66 men (39%). The median age of the patients was 74 years for the entire population with 76 years for women and 71 years for male patients. One hundred thirteen people (67%) had been previously hospitalised, and 5 person was a pensioner of a nursing home. 99 people (59%) were treated with antibiotics within 3 months before the first episode of infection. An average length of the hospital stay because of CDI was 11 days. One hundred thirty persons (77%) experienced only 1 episode whereas 38 people (23%) had more than 1 episode of infection. The person with the largest number of recurrences had 9 of them. CONCLUSIONS: The development of CDI is an increasing problem in a group of hospitalised persons, particularly of an old age. The general use of beta-lactam antibiotics is the cause of a larger number of infections with C. difficile. Vast majority of patients have had at least one typical risk factor of CDI.

Clostridium difficile infection in young people - 2 case reports.

Clostridium difficile infection (CDI) remains one of the most important healthcare-associated infections of the last two decades. The pathogen is a Gram-positive, toxin-producing, anaerobic, rod-shaped and sporeforming bacterium; it is ubiquitous in the human environment. Transmission occurs by the fecal-oral route. The consequence of the action of bacterial toxins is initially a local inflammatory reaction, which then goes into systemic inflammation. Clinical presentation is varied; some patients are asymptomatic, in symptomatic form the main symptom is diarrhea of varying severity, which is sometimes accompanied by acute abdominal pain, nausea, vomiting and high fever. Risk factors of CDI include prior antibiotic use, increasing age and recent hospitalisation. Extremely rarely does CDI occur in immunocompetent patients under 30 years of age, even if previously treated with an antibiotic. Here presented are two untypical cases of CDI development in the lower age group, in the presence of additional risk factors of comorbid gastrointestinal tract infections. Both developed infections are following recent infection - Salmonella enterididis in the first case and Salmonella typhi in the second case. Therefore, the article also contains basic principles for the diagnosis and treatment of Salmonella spp.

[Diagnostic difficulties in amebic liver abscess].

Amebiasis is a widespread parasitic infection caused by the human-specific protozoan Entamoeba histolytica (E.- histolytica). Tropical and subtropical regions with poor socioeconomic and sanitary conditions belong to endemic areas. The highest rates of E. histolytica infection are observed in India, Mexico, Africa, some parts ofCentral and South America. Up to 90% of infections remain asymptomatic, about 10% of patients develop amebic colitis. About 10% of symptomatic individuals may present with an extraintestinal manifestation, mostly amebic liver abscess (ALA). Clinical symptoms of ALA appear within 5 months after an exposition to E. histolytica cysts. Anamnesis revealing a travel to endemic area plays a crucial role in a diagnostic process, which is further supported by an physical examination, radiological findings, serology and parasitology test. The following article presents the difficulties which may occur when the ALA is suspected in a patient traveling from endemic areas.

n-3 and n-6 Fatty Acid Changes in the Erythrocyte Membranes of Patients with 658240251 Clostridium difficile Infection.

The implications of circulating essential fatty acids (FA) on the inflammatory risk profile and clinical outcome are still unclear. In order to gain a deeper understanding of the role of polyunsaturated fatty acids (PUFA) in the pathogenesis of acute infection, we analyzed the FA content in red blood cell (RBC) membranes of patients with Clostridium difficile infection (CDI) and controls. We prospectively studied 60 patients including 30 patients with CDI and 30 controls to assess lipid concentrations in erythrocyte membranes using gas chromatography. We observed a higher level of saturated fatty acids (SFA) in RBC membranes from patients with CDI. In patients with CDI, we also noticed a higher level of 20:4 n-6 FA and only a small amounts of C20:2n-6, C20:3n-6 FAs, arachidonic acid (AA) precursors, which suggest an intense inflammatory reaction in the organism during infection. We also noticed low levels of n-3 FA in the RBC membranes of patients infected with CDI. There is a deficit of n-3 FA in patients with CDI. n-3 FA are probably used during CDI as precursors of pro-resolving mediators that may indicate a therapeutic role of n-3 PUFAs in CDI. The changes in fatty acids in erythrocyte membranes during CDI alter their functions which may have an impact on the clinical outcome.

Transmitted HIV drug resistance in antiretroviral-treatment-naive patients from Poland differs by transmission category and subtype.

OBJECTIVES: The surveillance of HIV-transmitted drug resistance mutations (t-DRMs), including temporal trends across subtypes and exposure groups, remains a priority in the current management of the epidemic worldwide. METHODS: A cross-sectional analysis of 833 treatment-naive patients from 9 of 17 Polish HIV treatment centres. Partial pol sequences were used to analyse drug resistance with a general time reversible (GTR)-based maximum likelihood algorithm used for cluster/pair identification. Mutation frequencies and temporal trends were investigated. RESULTS: t-DRMs were observed in 9% of cases (5.8% for NRTI, 1.2% NNRTI and 2.0% PI mutations) and were more common among heterosexually infected (HET) individuals (13.4%) compared with MSM (8.3%, P = 0.03) or injection drug users (IDUs; 2.9%, P = 0.001) and in MSM compared with IDUs (P = 0.046). t-DRMs were more frequent in cases infected with the non-B variant (21.6%) compared with subtype B (6.6%, P < 0.001). With subtype B a higher mutation frequency was found in MSM compared with non-MSM cases (8.3% versus 1.8% for IDU + HET, P = 0.038), while non-B variants were associated with heterosexual exposure (30.4% for HET versus 4.8% for MSM, P = 0.019; versus 0 for IDU, P = 0.016). Trends in t-DRM frequencies were stable over time except for a decrease in NNRTI t-DRMs among MSM (P = 0.0662) and an NRTI t-DRM decrease in HET individuals (P = 0.077). With subtype B a higher frequency of sequence pairs/clusters in MSM (50.4%) was found compared with HET (P < 0.001) and IDUs (P = 0.015). CONCLUSIONS: Despite stable trends over time, patterns of t-DRMs differed notably between transmission categories and subtypes: subtype B was associated with MSM transmission and clustering while in non-B clades t-DRMs were more common and were associated with heterosexual infections.