Portable molecular diagnostic instruments in microbiology: current status.

BACKGROUND: Mobile microbiology is an evolving concept that has the potential to reduce morbidity and mortality associated with infectious diseases on a global level. Molecular methods used in the context of mobile microbiology ensure rapid and accurate aetiological diagnostics and allow timely initiation of clinical care. The great majority of published data regarding molecular diagnostics in mobile laboratories have focused on emerging viral infections and using laboratory-developed assays. Use of clinically validated and commercially available molecular diagnostic instruments in routine diagnostics of infectious diseases in mobile laboratories has received only limited attention in the field. OBJECTIVES: This review summarizes the suitability of a range of portable diagnostic molecular instruments for application in mobile laboratories by taking into account the instruments' analytical concepts, technical features and environmental requirements, as well as results of major validation studies. SOURCES: Data on technical features of selected portable instruments were mainly extracted from manufacturers' websites. Information on validation studies of various molecular assays developed for the selected instruments was extracted from peer-reviewed publications searched for through PubMed. CONTENT: Eight portable diagnostic molecular instruments (Alere q, GeneXpert Edge, GeneXpert Omni, Genedrive, PanNAT, Revogene, cobas Liat and ID Now) that are commercially available or in the launching stage are presented and evaluated in the context of the mobile microbiology concept, with particular emphasis on technical features and environmental requirements. Both the cobas Liat and the Alere i assays have been extensively validated in a variety of studies carried out in both adult and paediatric patients from various settings (ranging from primary care to emergency care departments in tertiary centres). Most studies showed comparable performance of cobas Liat and Alere i molecular assays with the standard-of-care in vitro diagnostics molecular assays routinely performed in dedicated/centralized molecular diagnostics laboratories. In addition, acceptable performance of Alere q and Genedrive instruments has been shown in implementations studies for early infant diagnosis of children born to human immunodeficiency virus-positive mothers and detection of hepatitis C virus RNA, respectively. Additional validation studies on existing (GeneXpert Edge, PanNAT, Revogene) and emerging (GeneXpert Omni) technologies are warranted. IMPLICATIONS: Several portable molecular diagnostic platforms reviewed are suitable for mobile microbiology applications. Further development in this field should be directed toward providing a broader range of assays per instrument, multiplexing, reducing the frequency of invalid results, and price cutting.

Primary resistance to integrase strand-transfer inhibitors in Europe.

OBJECTIVES: The objective of this study was to define the natural genotypic variation of the HIV-1 integrase gene across Europe for epidemiological surveillance of integrase strand-transfer inhibitor (InSTI) resistance. METHODS: This was a multicentre, cross-sectional study within the European SPREAD HIV resistance surveillance programme. A representative set of 300 samples was selected from 1950 naive HIV-positive subjects newly diagnosed in 2006-07. The prevalence of InSTI resistance was evaluated using quality-controlled baseline population sequencing of integrase. Signature raltegravir, elvitegravir and dolutegravir resistance mutations were defined according to the IAS-USA 2014 list. In addition, all integrase substitutions relative to HXB2 were identified, including those with a Stanford HIVdb score ≥ 10 to at least one InSTI. To rule out circulation of minority InSTI-resistant HIV, 65 samples were selected for 454 integrase sequencing. RESULTS: For the population sequencing analysis, 278 samples were retrieved and successfully analysed. No signature resistance mutations to any of the InSTIs were detected. Eleven (4%) subjects had mutations at resistance-associated positions with an HIVdb score ≥ 10. Of the 56 samples successfully analysed with 454 sequencing, no InSTI signature mutations were detected, whereas integrase substitutions with an HIVdb score ≥ 10 were found in 8 (14.3%) individuals. CONCLUSIONS: No signature InSTI-resistant variants were circulating in Europe before the introduction of InSTIs. However, polymorphisms contributing to InSTI resistance were not rare. As InSTI use becomes more widespread, continuous surveillance of primary InSTI resistance is warranted. These data will be key to modelling the kinetics of InSTI resistance transmission in Europe in the coming years.

Efficacy of tenofovir and efavirenz in combination with lamivudine or emtricitabine in antiretroviral-naive patients in Europe.

BACKGROUND: The combination of tenofovir and efavirenz with either lamivudine or emtricitabine (TELE) has proved to be highly effective in clinical trials for first-line treatment of HIV-1 infection. However, limited data are available on its efficacy in routine clinical practice. METHODS: A multicentre cohort study was performed in therapy-naive patients initiating ART with TELE before July 2009. Efficacy was studied using ITT (missing or switch = failure) and on-treatment (OT) analyses. Genotypic susceptibility scores (GSSs) were determined using the Stanford HIVdb algorithm. RESULTS: Efficacy analysis of 1608 patients showed virological suppression to <50 copies/mL at 48 weeks in 91.5% (OT) and 70.6% (ITT). Almost a quarter of all patients (22.9%) had discontinued TELE at week 48, mainly due to CNS toxicity. Virological failure within 48 weeks was rarely observed (3.3%, n = 53). In multilevel, multivariate analysis, infection with subtype B (P = 0.011), baseline CD4 count <200 cells/mm³ (P < 0.001), GSS <3 (P = 0.002) and use of lamivudine (P < 0.001) were associated with a higher risk of virological failure. After exclusion of patients using co-formulated compounds, virological failure was still more often observed with lamivudine. Following virological failure, three-quarters of patients switched to a PI-based regimen with GSS <3. After 1 year of second-line therapy, viral load was suppressed to <50 copies/mL in 73.5% (OT). CONCLUSIONS: In clinical practice, treatment failure on TELE regimens is relatively frequent due to toxicity. Virological failure is rare and more often observed with lamivudine than with emtricitabine. Following virological failure on TELE, PI-based second-line therapy was often successful despite GSS <3.

Serum apoptosis markers in HIV-infected patients with human herpesvirus type 8 and herpes simplex virus type 2 co-infection.

This study aimed to examine the influence of human herpesvirus type 8 (HHV-8) and herpes simplex virus type 2 (HSV-2) co-infections on apoptosis serum markers in human immunodeficiency virus (HIV)-infected patients. Sera from 110 HIV-infected and 59 HIV-uninfected individuals were analyzed for soluble Fas (sFas), sFas ligand (sFasL), caspase-8, and Bcl-2. The findings of HIV-infected patients with no co-infection (n = 37), HIV-infected patients with HHV-8 co-infection (n = 22), HIV-infected patients with HSV-2 co-infection (n = 51), and patients with HSV-2 co-infection and no HIV infection (n = 20) were compared to controls (reference group) with no HIV, HSV-2, and HHV-8 co-infections (n = 39). Soluble Fas and sFasL concentrations were the highest in HIV and HHV-8 co-infected patients (medians, 912.7 pg/ml and 74.3 pg/mL, respectively). No difference in caspase-8 concentrations was found, whereas Bcl-2 concentrations were the highest in HIV and HHV-8 co-infected individuals. Older age was associated with higher sFas (p < 0.001) and lower sFasL (p = 0.04) concentrations. In a robust regression model adjusted for age, the log-transformed sFas concentrations were significantly lower in HIV-infected patients with no co-infections (ß = -0.244; p < 0.001) and higher in HIV and HHV-8 co-infected patients (ß = 0.216; p = 0.012) compared to the reference group. Soluble FasL was significantly lower in HIV-infected patients with no co-infections (ß = -0.284; p = 0.005) and in HIV-infected patients with HSV-2 co-infection (ß = -0.381; p < 0.001) compared to the reference group. Soluble FasL was also higher in HIV and HHV-8 co-infected patients compared to controls (ß = 0.248; p = 0.036). Our results suggest that HHV-8 and HSV-2 may have a significant effect on Fas-FasL-mediated apoptosis in HIV-1 patients. HHV-8 upregulates while HSV-2 downregulates sFas and sFasL.

Comparison of clinical symptoms scored according to the National Institutes of Health chronic prostatitis symptoms index and assessment of antimicrobial treatment in patients with chronic prostatitis syndrome.

We examined a total of 194 patients over 18 years of age with chronic prostatitis syndrome and no evidence of structural or functional lower genitourinary tract abnormalities. The following data were obtained for each patient: clinical history--the severity of chronic prostatitis symptoms scored by a Croatian translation of the NiH CPSI questionnaire, clinical status including digitorectal examination, urethral swab specimens, and selective samples of urine and expressed prostatic secretion, according to the 4-glass localization test (meares and Stamey localization technique). Patients were treated orally with antimicrobial agents in doses and duration according to clinical practice in Croatia. An infectious etiology was determined in 169 (87%) patients. Chlamydia trachomatis was the causative pathogen in 38 (20%), Trichomonas vaginalis in 35 (18%), Enterococcus in 36 (19%) and Escherichia coli in 35 (18%) patients. In the remaining 25 patients the following causative pathogens were found: Ureaplasma urealyticum, Proteus mirabilis, Klebsiella pneumoniae, Streptococcus agalactiae and Pseudomonas aeruginosa. Comparison of symptoms scores and effect on quality of life has shown that the most severe clinical presentation of disease was recorded in patients with chronic bacterial prostatitis caused by E. coli and Enterococcus (p<0.001). Clinical success was paralleled by bacteriological eradication in chronic bacterial prostatitis caused by C. trachomatis, Enterococcus and E. coli (kappa >0.2<0.5), but not in inflammatory chronic pelvic pain syndrome caused by T. vaginalis.

The significance of Chlamydia trachomatis in urethritis and prostatitis - differences in therapeutic approach - Croatian experience.

We examined a total of 1014 patients over 18 years of age; 252 with urethritis and 762 with chronic prostatitis syndrome. the mean age of patients with urethritis was 32.7 and with prostatitis syndrome 37.6 years. Clinical symptoms of urethritis were present from a few days to several months. in patients with chronic prostatitis syndrome, symptoms were present for at least 3 months. Chlamydia trachomatis alone was confirmed in 26 (10%) and in combination with Ureaplasma urealyticum in 6 (2%) patients with urethritis. in 171 (68%) patients with urethritis neither C. trachomatis nor U. urealyticum or Mycoplasma hominis were found. C. trachomatis alone was confirmed in 70 (9%), and in combination with other microorganisms in 7 (1%) patients with chronic prostatitis syndrome. in Croatia, the frequency of chronic chlamydial prostatitis has not significantly changed in the last 10 years, while the frequency of infections among adolescents decreased. the recommended regimen for acute chlamydial urethritis in Croatia is azithromycin 1.0 g as a single dose, and a total dose of 4-4.5 g azithromycin for chronic chlamydial prostatitis.

Increased numbers of CD38 molecules on bright CD8+ T lymphocytes in infectious mononucleosis caused by Epstein-Barr virus infection.

The aim of this study was to quantify the expression of CD38 on CD8+ T lymphocytes of patients with infectious mononucleosis (IM) caused by Epstein-Barr virus (EBV) and cytomegalovirus (CMV). CD38 quantification technique chosen for this study was based on the enumeration of CD38 antibody binding sites in comparison to the quantification standards rather than determining relative fluorescence, which is difficult to standardize. The study enrolled 19 patients with typical clinical and laboratory parameters compatible with EBV-induced IM as well as 10 patients with atypical clinical presentation of this disease. Furthermore, CD38 expression was analysed in a group of 13 patients with IM caused by CMV infection. CD38 quantification was performed within 6 days of the presentation of symptoms. All three groups of IM patients showed a statistically significant increase in the number of anti-CD38 antibody binding sites (which correspond to the number of CD38 molecules) on bright CD8+ T lymphocytes compared to healthy controls. The numbers of CD38 molecules expressed on CD8+ T lymphocytes did not differ significantly between IM patients with typical and atypical clinical presentation of the disease. Patients with CMV-induced IM had significantly lower numbers of CD38 molecules expressed on CD8+ T lymphocytes. Therefore, we conclude that CD38 quantification could be helpful in differential diagnostics of IM cases with atypical clinical presentation.