Development and Application of a Broadly Sensitive Dried-Blood-Spot-Based Genotyping Assay for Global Surveillance of HIV-1 Drug Resistance

As antiretroviral therapy (ART) is scaled up in resource-limited countries, surveillance for HIV drug resistance (DR) is vital to ensure sustained effectiveness of first-line ART. We have developed and applied a broadly sensitive dried-blood-spot (DBS)-based genotyping assay for surveillance of HIV-1 DR in international settings. In 2005 and 2006, 171 DBS samples were collected under field conditions from newly diagnosed HIV-1-infected individuals from Malawi (n = 58), Tanzania (n = 60), and China (n =53). In addition, 30 DBS and 40 plasma specimens collected from ART patients in China and Cameroon, respectively, were also tested. Of the 171 DBS analyzed at the protease and RT regions, 149 (87.1%) could be genotyped, including 49 (81.7%) from Tanzania, 47 (88.7%) from China, and 53 (91.4%) from Malawi. Among the 70 ART patient samples analyzed, 100% (30/30) of the Chinese DBS and 90% (36/40) of the Cameroonian plasma specimens were genotyped, including 8 samples with a viral load of <400 copies/ml. The results of phylogenetic analyses indicated that the subtype, circulating recombinant form (CRF), and unique recombinant form (URF) distribution was as follows: 73 strains were subtype C (34%), 37 were subtype B (17.2%), 24 each were CRF01_AE or CRF02_AG (11.2% each), 22 were subtype A1 (10.2%), and 9 were unclassifiable (UC) (4.2%). The remaining samples were minor strains comprised of 6 that were CRF07_BC (2.8%), 5 that were CRF10_CD (2.3%), 3 each that were URF_A1C and CRF08_BC (1.4%), 2 each that were G, URF_BC, and URF_D/UC (0.9%), and 1 each that were subtype F1, subtype F2, and URF_A1D (0.5%). Our results indicate that this broadly sensitive genotyping assay can be used to genotype DBS collected from areas with diverse HIV-1 group M subtypes and CRFs. Thus, the assay is likely to become a useful screening tool in the global resistance surveillance and monitoring of HIV-1 where multiple subtypes and CRFs are found.Filter papers have been useful for the collection, storage, and testing of blood specimens in the diagnostic screening of metabolic and inherited disorders in newborn babies in the United States for many years (13, 14). More recently, many resource-limited countries have been using them for HIV-related molecular assays, including early infant diagnosis using Roche Amplicor HIV-1 DNA PCR testing (supported by the U.S. President''s Emergency Plan for AIDS Relief [PEPFAR]) (31), real-time PCR-based HIV-1 diagnosis (22, 27) and viral load measurement (6, 17, 18, 20, 26), and HIV-1 drug resistance (DR) genotyping (5, 7, 8, 12, 16, 19, 23-25, 28, 30, 35-37).Dried blood spots (DBS) offer several advantages over conventional plasma or serum for sample collection and storage. First, DBS circumvent the need for phlebotomy and reduce the risk of needle-stick-related HIV exposure when they are collected using finger or heel stick. Second, they do not require cold-chain transportation of specimens from collection sites to the testing laboratories; thus, they can be transported using standard postal services, resulting in the reduction of cost for storage and transportation when dried and processed appropriately. Overall, DBS provide resource-limited countries with opportunities for sustainable laboratory services and surveillance programs for HIV diagnosis and DR genotyping. Very few studies have been conducted using DBS collected under real field conditions. Furthermore, the DR genotyping assays used were not evaluated for multiple HIV-1 subtypes and circulating recombinant forms (CRFs) (5, 16, 19, 23, 25, 37).The aim of the current study was to determine the performance of a broadly sensitive genotyping assay for detecting multiple HIV-1 group M subtypes and CRFs that cocirculate in different countries, using DBS collected under field conditions.     

Low Frequency of CXCR4-Using Viruses in Patients at the Time of Primary Non-Subtype-B HIV-1 Infection

We used genotypic and phenotypic assays to estimate the frequency of X4/DM viruses in 131 patients infected with non-subtype-B viruses at the time of primary HIV-1 infection (PHI). All patients were enrolled in the French PRIMO Cohort from 1996 to 2007. Most strains belonged to CRF02_AG (51.1%) and subtype A (14.5%). Sixteen viruses (12.2%) were classified as CXCR4 tropic (“X4 strains”) by the combined criteria of amino acids 11 and 25 of the V3 loop (11/25) and net charge rules and/or the SVMgeno2pheno_10% algorithm: 6 strains by the combined genotypic rule, 7 by the SVMgeno2pheno_10% algorithm, and 3, clustering in subtype D, by both algorithms. However, only one strain (0.8%), belonging to subtype A, was defined as a dual-tropic (DM) virus by the phenotypic assay. The 67 CRF02_AG strains included 2 classified as X4 strains by the combined genotypic rule (3%) and 2 others classified as X4 strains by SVMgeno2pheno_10% (3%), but none of these 4 strains was an X4 or DM strain according to the phenotypic assay. These results suggest that the cellular virus reservoir was established with X4 strains in very few non-subtype-B-infected patients at the time of PHI. Genotypic predictions can overestimate the proportion of non-subtype-B X4 viruses at PHI.Human immunodeficiency virus type 1 (HIV-1) can be characterized by the host chemokine coreceptor that it uses to enter CD4-expressing cells. HIV-1 variants usually bind to the CCR5 chemokine coreceptor early in the course of disease. These are “R5” viruses (3, 31, 48). Viruses that use another chemokine coreceptor, CXCR4, are “X4” viruses, and they emerge later in HIV infection. They can account for up to 40 to 50% of all viruses in heavily treated patients with advanced disease (1, 32). The presence of X4 viruses has been associated with accelerated disease progression and a precipitous loss of CD4 T cells (27, 29, 40). A recent Swiss study suggested that the presence of X4 strains and the X4-specific virus load strongly predict clinical disease progression during combined antiretroviral therapy (cART), in addition to the CD4 T-cell count or viral load (44). This potential correlation between virus tropism and disease progression has important clinical implications. The development of coreceptor CCR5 antagonists for treating retroviruses and the lack of a virological response by patients infected with X4 or dual/mixed (X4/DM) viruses have increased the need to determine HIV-1 tropism.Recent studies have found the frequency of X4/DM dual-tropic strains in plasma samples from recently infected patients in the United States and Spain to be from 3.2% to 17.5% (14, 15, 16). Similarly, we found 15.9% (95% confidence interval [CI], 12.3% to 19.5%) strains of X4/DM viruses in 390 HIV-1 subtype B-infected patients diagnosed at the time of primary HIV-1 infection (PHI) in France from 1996 to 2007 (18).One of the major challenges of determining tropism is to select the best method for identifying coreceptor usage. HIV coreceptor usage is most commonly determined with a recombinant phenotype assay in clinical studies (28, 45). Bioinformatic tools based on the virus genotype may also be able to predict coreceptor usage. They are faster, less expensive, and more suitable for studies of a large number of patients than are phenotypic recombinant assays. Each available genotypic test is adequately specific but not very sensitive for detecting X4/DM or X4 variants. An overall concordance of 71.2 to 92% between genotypic and phenotypic assays has been reported (8, 15, 37, 41). However, most of these studies included HIV-1 subtype B strains. Genotypic algorithms may not be suitable for predicting the tropism of non-subtype-B HIV-1 strains (20). Two recent studies demonstrated that genotypic tests performed well for predicting the coreceptor usage of CRF02_AG and subtype C strains (36, 38), but no study has examined the correlation between genotypic and phenotypic tests for predicting the tropism of non-subtype-B HIV-1 at the time of PHI. The French PRIMO Cohort contained a large proportion of patients infected with a non-subtype-B virus (25.5% in 2005 to 2006) (6).We have therefore estimated the frequency of X4/DM viruses in 131 patients infected with non-subtype-B viruses at the time of PHI. All of them were enrolled in the French PRIMO Cohort from 1996 to 2007. We also studied the concordance between genotypic and phenotypic assays for predicting the tropism of non-subtype-B viruses in these patients.     

Evaluation of the Abbott m2000 RealTime Human Immunodeficiency Virus Type 1 (HIV-1) Assay for HIV Load Monitoring in South Africa Compared to the Roche Cobas AmpliPrep-Cobas Amplicor,Roche Cobas AmpliPrep-Cobas TaqMan HIV-1, and BioMerieux NucliSENS EasyQ HIV-1 Assays

The implementation of antiretroviral therapy demands the need for increased access to viral load (VL) monitoring. Newer real-time VL testing technologies are faster and have larger dynamic ranges and fully automated extraction to benefit higher throughputs in resource-poor environments. The Abbott RealTime human immunodeficiency virus type 1 (HIV-1) assay was evaluated as a new option for testing for HIV-1 subtype C in South Africa, and its performance was compared to the performance of existing assays (the Cobas AmpliPrep-Cobas TaqMan HIV-1, version 1, assay; the AmpliPrep-Cobas Monitor standard HIV-1 assay; and the NucliSENS EasyQ-EasyMag HIV-1 assay) in a high-throughput laboratory. The total precision of the RealTime HIV-1 assay was acceptable over all viral load ranges. This assay compared most favorably with the Cobas AmpliPrep-Cobas TaqMan HIV-1 assay (R² = 0.904), with a low standard deviation of difference being detected (0.323 copies/ml). The bias against comparator assays ranged from −0.001 copies/ml to −0.228 copies/ml. Variability in the reporting of VLs for a 20-member subtype panel compared to the variability of other assays was noted with subtypes G and CRF02-AG. The RealTime HIV-1 assay can test 93 samples per day with minimal manual preparation, less staff, and the minimization of contamination through automation. This assay is suitable for HIV-1 subtype C VL quantification in South Africa.South Africa bears a huge burden of human immunodeficiency virus (HIV) infection and has an estimated 5.5 million infected individuals (9). In September 2007, approximately 329,000 individuals received antiretroviral (ARV) treatment as part of the national program (24a, 40). Globally, viral load (VL) testing has been used to monitor treatment, determine the prognosis and the risk of disease progression, and identify treatment failures (47). South African treatment guidelines use VL results to determine the time for the switch from a first-line to a second-line drug regimen (24). Work from the region has demonstrated that the use of VL monitoring together with targeted adherence approaches results in significant conservation of the first-line drug regimen (2). In addition, it has been demonstrated that the switching of therapy on the basis of clinical and immunological criteria alone results in the development of more complex resistance profiles (42), thus reinforcing the value of VL monitoring. Initial WHO guidelines for ARV treatment in resource-poor settings referred to the use of VL monitoring as optional (45). There has been a recent shift in thinking, with the revised guidelines referring to VL testing as being desirable in resource-limited settings (11) and with greater international efforts being focused on improving access to virological testing.Nucleic acid amplification technologies are used for VL testing in most settings and are based on either signal amplification (27) or target amplification (20) methodological approaches. The trend now is toward the use of the real-time detection of amplicons (43), which simply means that detection is done as the product accumulates during the exponential phase of the reaction. Newer real-time technology options are faster and have higher throughputs, larger dynamic ranges, and fully automated extraction steps (43). To ensure that the South African National Health Laboratory Service can cope with the high volumes of samples anticipated as the treatment program matures, several automated, real-time monitoring assays have been explored in country.In the study described here, we investigated and compared the performance of the Abbott Diagnostics (Abbott Park, IL) RealTime HIV type 1 (HIV-1) assay (abbreviated RealTime HIV-1) on the m2000sp/m2000rt automated extraction platform (Abbott Molecular Inc., Des Plaines, IL) to the performance of the Cobas AmpliPrep-Cobas TaqMan HIV-1, version 1, assay (abbreviated CAP/CTM HIV-1; F. Hoffmann-La Roche, Diagnostics Division, Basel, Switzerland); the NucliSENS EasyQ-EasyMag HIV-1, version 1.1, assay (abbreviated NucliSENS HIV-1; bioMérieux, Boxtel, The Netherlands); and the AmpliPrep-Cobas Monitor standard HIV-1 assay (abbreviated CAP/CA HIV-1), assays which are already established within the South African laboratory setting (32, 33).The first three assays mentioned above use real-time platforms with fluorescence detection systems, and CAP/CA HIV-1 is based on endpoint detection. Previous publications reported on the performance of CAP/CTM HIV-1 (12, 17, 25, 26, 28) and NucliSENS HIV-1 (8, 13, 32, 33). RealTime HIV-1 has previously been validated against the Roche HIV-1 Monitor assay (which uses a manual RNA extraction step) and CAP/CTM HIV-1 with the m1000 system, which is less automated than the m2000sp system used in this study (6, 15). Other studies have compared RealTime HIV-1 to the Roche Cobas Monitor assay for VL testing for HIV-1 and hepatitis C virus (29, 37, 38). Swanson and colleagues (2006) compared RealTime HIV-1 with the fully automated m2000 system, CAP/CA HIV-1 (version 1.5), the Versant HIV-1 RNA (version 3.0) system, and the Abbott LCx HIV-1 RNA quantitative system (38). A recent study has also compared RealTime HIV-1 to NucliSENS HIV-1 for the detection of the HIV-1 clades prevalent in China (46). Although those reports described the performance of the Abbott VL assay alone or in comparison to that of several existing VL assays for the detection of different subtypes, the study presented here is the first evaluation of RealTime HIV-1, NucliSENS HIV-1, CAP/CTM HIV-1, and CAP/CA HIV-1 predominantly for the detection of HIV-1 subtype C in South Africa.     

Variation in Susceptibility of Bloodstream Isolates of Candida glabrata to Fluconazole According to Patient Age and Geographic Location in the United States in 2001 to 2007

We examined the susceptibilities to fluconazole of 642 bloodstream infection (BSI) isolates of Candida glabrata and grouped the isolates by patient age and geographic location within the United States. Susceptibility of C. glabrata to fluconazole was lowest in the northeast region (46%) and was highest in the west (76%). The frequencies of isolation and of fluconazole resistance among C. glabrata BSI isolates were higher in the present study (years 2001 to 2007) than in a previous study conducted from 1992 to 2001. Whereas the frequency of C. glabrata increased with patient age, the rate of fluconazole resistance declined. The oldest age group (≥80 years) had the highest proportion of BSI isolates that were C. glabrata (32%) and the lowest rate of fluconazole resistance (5%).Candidemia is without question the most important of the invasive mycoses (6, 33, 35, 61, 65, 68, 78, 86, 88). Treatment of candidemia over the past 20 years has been enhanced considerably by the introduction of fluconazole in 1990 (7, 10, 15, 28, 29, 31, 40, 56-58, 61, 86, 90). Because of its widespread usage, concern about the development of fluconazole resistance among Candida spp. abounds (2, 6, 14, 32, 47, 53, 55, 56, 59, 60, 62, 80, 86). Despite these concerns, fluconazole resistance is relatively uncommon among most species of Candida causing bloodstream infections (BSI) (5, 6, 22, 24, 33, 42, 54, 56, 65, 68, 71, 86). The exception to this statement is Candida glabrata, of which more than 10% of BSI isolates may be highly resistant (MIC ≥ 64 μg/ml) to fluconazole (6, 9, 15, 23, 30, 32, 36, 63-65, 71, 87, 91). Suboptimal fluconazole dosing practices (low dose [<400 mg/day] and poor indications) may lead to an increased frequency of isolation of C. glabrata as an etiological agent of candidemia in hospitalized patients (6, 17, 29, 32, 35, 41, 47, 55, 60, 68, 85) and to increased fluconazole (and other azole) resistance secondary to induction of CDR efflux pumps (2, 11, 13, 16, 43, 47, 50, 55, 69, 77, 83, 84) and may adversely affect the survival of treated patients (7, 10, 29, 40, 59, 90). Among the various Candida species, C. glabrata alone has increased as a cause of BSI in U.S. intensive care units since 1993 (89). Within the United States, the proportion of fungemias due to C. glabrata has been shown to vary from 11% to 37% across the different regions (west, midwest, northeast, and south) of the country (63, 65) and from <10% to >30% within single institutions over the course of several years (9, 48). It has been shown that the prevalence of C. glabrata as a cause of BSI is potentially related to many disparate factors in addition to fluconazole exposure, including geographic characteristics (3, 6, 63-65, 71, 88), patient age (5, 6, 25, 35, 41, 42, 48, 63, 82, 92), and other characteristics of the patient population studied (1, 32, 35, 51). Because C. glabrata is relatively resistant to fluconazole, the frequency with which it causes BSI has important implications for therapy (21, 29, 32, 40, 41, 45, 56, 57, 59, 80, 81, 86, 90).Previously, we examined the susceptibilities to fluconazole of 559 BSI isolates of C. glabrata and grouped the isolates by patient age and geographic location within the United States over the time period from 1992 to 2001 (63). In the present study we build upon this experience and report the fluconazole susceptibilities of 642 BSI isolates of C. glabrata collected from sentinel surveillance sites throughout the United States for the time period from 2001 through 2007 and stratify the results by geographic region and patient age. The activities of voriconazole and the echinocandins against this contemporary collection of C. glabrata isolates are also reported.     

Dried-Plasma Transport Using a Novel Matrix and Collection System for Human Immunodeficiency Virus and Hepatitis C Virus Virologic Testing

A novel method for the collection and transportation of dried-blood-plasma samples, SampleTanker (ST), was developed and compared to standard shipping protocols for frozen-plasma specimens containing human immunodeficiency virus type 1 (HIV-1) and/or hepatitis C virus (HCV). Matched frozen and dried 1-ml EDTA-containing plasma samples were collected and analyzed by several molecular-based virologic assays. After addition of 1.175 ml of reconstitution buffer, 1.035 ml of dried plasma was recovered. Mean intra-assay variances were 0.05, 0.05, and 0.06 log₁₀ copies/ml for the Versant, Amplicor, and NucliSens QT HIV-1 load assays, respectively (P, not significant). However, mean HIV-1 viral load was consistently reduced in dried samples by 0.32 to 0.51 log₁₀ copies/ml, depending on assay type (P < 0.05). Infectious HIV-1 was not recovered from dried ST plasma. There was no significant difference in HIV-1 viral load results obtained using ST after 8 weeks of storage at ambient temperature. Compared to frozen plasma, HIV-1 genotypic results were >99% concordant at the nucleotide and amino acid levels, as well as for resistance-associated mutations. We further demonstrated successful detection of multiple analytes, including HIV-1 viral load, HIV-1 antiretroviral resistance genotype, and HCV genotype, from a single ST unit. Dried plasma collected with ST yielded comparable results to frozen samples for multiple-analyte clinical testing. As such, ST could be a useful alternative for virologic tests and clinical trials worldwide by significantly diminishing transportation cost and the sample volume restrictions associated with dried-blood-spot technology.Diagnostic and therapeutic monitoring assays for human immunodeficiency virus type 1 (HIV-1) and hepatitis C virus (HCV) infections are useful surrogate markers in the management and treatment of these infections (29). In addition, HIV-1 and HCV viral load, HCV genotype, and HIV-1 resistance testing are strongly correlated with response to antiviral therapy (27). Currently, blood tests which utilize HIV-1 and HCV viral nucleic acid require plasma separation and freezing at −70°C or greater within a few hours of collection. Most clinical samples and those associated with clinical trials require overnight shipment to reference laboratories under frozen conditions, which is expensive and cumbersome. In addition, the processing, storage, and transportation requirements for these assays limit their utility and accessibility in resource-poor environments.The use of dried blood, serum, or plasma transport medium for viral diagnostics and therapeutic monitoring would offer several advantages over current sample collection and transportation requirements. The utility of using a dry blood spot (DBS) on filter paper has been a well-established whole-blood collection method for congenital and inherited metabolic neonatal screening programs and was demonstrated to be effective in the collection of serum containing thyroid hormone several decades ago (16). The utility of DBSs was reported early in the AIDS epidemic for HIV-1 antibody detection (12) and continues to be evaluated for antibodies to various subtypes and for p24 antigen testing (4, 17, 18, 21, 31). The DBS has also been used for detection of antibodies against other viral infections such as HCV, human T-cell leukemia virus type 1, and hepatitis B surface antigen (10), as well as HIV-1 proviral DNA in peripheral blood mononuclear cells (PBMCs) and HIV and HCV RNA viral load using PCR (6, 7, 13, 14, 28, 30). Dried-blood samples have been used for HIV-1 subtype analysis and CD4⁺ T-cell and plasma antiretroviral drug concentration quantification (8, 19, 24, 26, 33), and antiretroviral (ARV) genotypic resistance testing has been demonstrated (5, 22, 32, 35). Phenotypic ARV resistance testing or the feasibility to assay multiple viral nucleic acid analytes simultaneously from a single sample has not been described.In this study, we describe a novel, non-paper-based matrix sample collection and transportation device called SampleTanker (ST), which was used to collect dried blood plasma for virologic testing. We evaluated the feasibility, precision, and short-term stability of using ST as a transportation method for shipping specimens at ambient temperature and for subsequent individual or simultaneous testing using HIV-1 viral load, HIV genotypic resistance testing, and HCV genotyping assays.     

Overestimation of Human Immunodeficiency Virus Type 1 Load Caused by the Presence of Cells in Plasma from Plasma Preparation Tubes

The human immunodeficiency virus type 1 (HIV-1) load is an important marker of disease progression and treatment efficacy in patients with HIV-1 infection. In recent years, an increase in the number of samples with detectable HIV-1 RNA has been reported among patients with previously suppressed viral loads, affecting clinical patient care and leading to repeat measurements of viral load and drug resistance. This rise seems to have coincided with the increased use of plasma preparation tubes (PPTs) for sample collection, and we have aimed to explain why PPTs might yield elevated HIV-1 RNA levels. The impacts of different sample-processing procedures on HIV-1 RNA levels were compared retrospectively. Prospectively, the presence of different cells and cell-associated HIV-1 nucleic acids in paired plasma samples from PPTs centrifuged before (PPT1) and after (PPT2) transportation to the laboratory was compared. A retrospective analysis of 4,049 patient samples with <1,000 HIV-1 RNA copies/ml showed elevated HIV-1 RNA levels in plasma from PPT1 compared with the levels from PPT2 and standard EDTA-containing tubes. Prospective data revealed cell-associated HIV-1 nucleic acids and abundant blood cells in plasma from PPT1 but not from the corresponding PPT2. The levels of HIV-1 RNA correlated with the lymphocyte counts in plasma in PPT1. Cells could be removed by the recentrifugation of PPT1 before analysis. In conclusion, the transportation of PPTs after centrifugation may render cells in the plasma fraction containing cell-associated HIV-1 nucleic acids that contribute significantly to the HIV-1 RNA copy numbers in patients with low viral loads.Quantification of human immunodeficiency virus type 1 (HIV-1) RNA in plasma is an essential tool in the clinical management of patients with HIV-1 infection and is used to monitor HIV-1 disease progression and the response to antiretroviral therapy (ART) (9, 12, 14, 15). It is also an important end point in most HIV-1 treatment clinical trials.Although intermittent low-level viremia is often reported among patients receiving seemingly effective ART (4, 13, 16, 23), the unexpected detection of HIV-1 RNA might have a clinical impact and lead to repeat quantification of HIV-1 RNA and testing for drug resistance. In recent years, several HIV-1 clinics and laboratories have reported an increased number of cases of detectable HIV-1 RNA in patients with previous suppressed viral loads, raising concerns about drug resistance and virologic failure (8, 17, 20). At the Oslo University Hospital, Ullevål, we have also experienced an unusual increase in the proportion of plasma samples with HIV-1 RNA levels in the range of 40 to 1,000 copies/ml. This seems to have coincided with a change in the routines for plasma sample collection, and here we present data providing an explanation for this phenomenon.It is recommended that blood samples for HIV-1 RNA quantification be collected in tubes with EDTA as an anticoagulant (3, 12). Standard EDTA-containing tubes require transfer of the plasma to a secondary tube within 6 h after sample collection to reduce the risk of RNA degradation (3). This is often inconvenient in a patient clinic, and as an alternative, plasma preparation tubes (PPTs; Becton Dickinson [BD], Franklin Lakes, NJ) are increasingly often used. Upon centrifugation of the PPTs, a gel barrier separates the plasma from most of the cellular elements, theoretically allowing transportation of the sample in the primary tube.The collection of samples in PPTs from patients receiving effective ART is reported to yield increased levels of HIV-1 RNA compared with the levels in plasma collected in standard EDTA-containing tubes (5, 6, 8, 17, 18, 20, 21). However, the reason for this discrepancy has remained unclear. Our data confirm the previous findings that PPTs generate significantly higher HIV-1 RNA levels in samples from patients with low-level viremia. Additionally, we have shown that the source of this overestimation is cell-associated HIV-1 nucleic acids. These findings necessitate a reconsideration of low-level viral load results in plasma obtained from PPTs not handled with care after centrifugation. Furthermore, we present a simple procedure that will circumvent the problem.     

Hag Mediates Adherence of Moraxella catarrhalis to Ciliated Human Airway Cells

Moraxella catarrhalis is a human pathogen causing otitis media in infants and respiratory infections in adults, particularly patients with chronic obstructive pulmonary disease. The surface protein Hag (also designated MID) has previously been shown to be a key adherence factor for several epithelial cell lines relevant to pathogenesis by M. catarrhalis, including NCIH292 lung cells, middle ear cells, and A549 type II pneumocytes. In this study, we demonstrate that Hag mediates adherence to air-liquid interface cultures of normal human bronchial epithelium (NHBE) exhibiting mucociliary activity. Immunofluorescent staining and laser scanning confocal microscopy experiments demonstrated that the M. catarrhalis wild-type isolates O35E, O12E, TTA37, V1171, and McGHS1 bind principally to ciliated NHBE cells and that their corresponding hag mutant strains no longer associate with cilia. The hag gene product of M. catarrhalis isolate O35E was expressed in the heterologous genetic background of a nonadherent Haemophilus influenzae strain, and quantitative assays revealed that the adherence of these recombinant bacteria to NHBE cultures was increased 27-fold. These experiments conclusively demonstrate that the hag gene product is responsible for the previously unidentified tropism of M. catarrhalis for ciliated NHBE cells.Moraxella catarrhalis is a gram-negative pathogen of the middle ear and lower respiratory tract (29, 40, 51, 52, 69, 78). The organism is responsible for ∼15% of bacterial otitis media cases in children and up to 10% of infectious exacerbations in patients with chronic obstructive pulmonary disease (COPD). The cost of treating these ailments places a large financial burden on the health care system, adding up to well over $10 billion per annum in the United States alone (29, 40, 52, 95, 97). In recent years, M. catarrhalis has also been increasingly associated with infections such as bronchitis, conjunctivitis, sinusitis, bacteremia, pneumonia, meningitis, pericarditis, and endocarditis (3, 12, 13, 17-19, 24, 25, 27, 51, 67, 70, 72, 92, 99, 102-104). Therefore, the organism is emerging as an important health problem.M. catarrhalis infections are a matter of concern due to high carriage rates in children, the lack of a preventative vaccine, and the rapid emergence of antibiotic resistance in clinical isolates. Virtually all M. catarrhalis strains are resistant to β-lactams (34, 47, 48, 50, 53, 65, 81, 84). The genes specifying this resistance appear to be gram positive in origin (14, 15), suggesting that the organism could acquire genes conferring resistance to other antibiotics via horizontal transfer. Carriage rates as high as 81.6% have been reported for children (39, 104). In one study, Faden and colleagues analyzed the nasopharynx of 120 children over a 2-year period and showed that 77.5% of these patients became colonized by M. catarrhalis (35). These investigators also observed a direct relationship between the development of otitis media and the frequency of colonization. This high carriage rate, coupled with the emergence of antibiotic resistance, suggests that M. catarrhalis infections may become more prevalent and difficult to treat. This emphasizes the need to study pathogenesis by this bacterium in order to identify vaccine candidates and new targets for therapeutic approaches.One key aspect of pathogenesis by most infectious agents is adherence to mucosal surfaces, because it leads to colonization of the host (11, 16, 83, 93). Crucial to this process are surface proteins termed adhesins, which mediate the binding of microorganisms to human cells and are potential targets for vaccine development. M. catarrhalis has been shown to express several adhesins, namely UspA1 (20, 21, 59, 60, 77, 98), UspA2H (59, 75), Hag (also designated MID) (22, 23, 37, 42, 66), OMPCD (4, 41), McaP (61, 100), and a type 4 pilus (63, 64), as well as the filamentous hemagglutinin-like proteins MhaB1, MhaB2, MchA1, and MchA2 (7, 79). Each of these adhesins was characterized by demonstrating a decrease in the adherence of mutant strains to a variety of human-derived epithelial cell lines, including A549 type II pneumocytes and Chang conjunctival, NCIH292 lung mucoepidermoid, HEp2 laryngeal, and 16HBE14o-polarized bronchial cells. Although all of these cell types are relevant to the diseases caused by M. catarrhalis, they lack important aspects of the pathogen-targeted mucosa, such as the features of cilia and mucociliary activity. The ciliated cells of the respiratory tract and other mucosal membranes keep secretions moving out of the body so as to assist in preventing colonization by invading microbial pathogens (10, 26, 71, 91). Given this critical role in host defense, it is interesting to note that a few bacterial pathogens target ciliated cells for adherence, including Actinobacillus pleuropneumoniae (32), Pseudomonas aeruginosa (38, 108), Mycoplasma pneumoniae (58), Mycoplasma hyopneumoniae (44, 45), and Bordetella species (5, 62, 85, 101).In the present study, M. catarrhalis is shown to specifically bind to ciliated cells of a normal human bronchial epithelium (NHBE) culture exhibiting mucociliary activity. This tropism was found to be conserved among isolates, and analysis of mutants revealed a direct role for the adhesin Hag in binding to ciliated airway cells.     

Acanthamoeba culbertsoni Elicits Soluble Factors That Exert Anti-Microglial Cell Activity

Acanthamoeba culbertsoni is an opportunistic pathogen that causes granulomatous amoebic encephalitis (GAE), a chronic and often fatal disease of the central nervous system (CNS). A hallmark of GAE is the formation of granulomas around the amoebae. These cellular aggregates consist of microglia, macrophages, lymphocytes, and neutrophils, which produce a myriad of proinflammatory soluble factors. In the present study, it is demonstrated that A. culbertsoni secretes serine peptidases that degrade chemokines and cytokines produced by a mouse microglial cell line (BV-2 cells). Furthermore, soluble factors present in cocultures of A. culbertsoni and BV-2 cells, as well as in cocultures of A. culbertsoni and primary neonatal rat cerebral cortex microglia, induced apoptosis of these macrophage-like cells. Collectively, the results indicate that A. culbertsoni can apply a multiplicity of cell contact-independent modes to target macrophage-like cells that exert antiamoeba activities in the CNS.Acanthamoeba culbertsoni belongs to a group of free-living amoebae, such as Balamuthia mandrillaris, Naegleria fowleri, and Sappinia pedata, that can cause disease in humans (46, 56). Acanthamoeba spp. are found worldwide and have been isolated from a variety of environmental sources, including air, soil, dust, tap water, freshwater, seawater, swimming pools, air conditioning units, and contaminated contact lenses (30). Trophozoites feed on bacteria and algae and represent the infective form (47, 56). However, under unfavorable environmental conditions, such as extreme changes in temperature or pH, trophozoites transform into a double-walled, round cyst (22, 45).Acanthamoeba spp. cause an infection of the eye known as amoebic keratitis (AK), an infection of the skin referred to as cutaneous acanthamoebiasis, and a chronic and slowly progressing disease of the central nervous system (CNS) known as granulomatous amoebic encephalitis (GAE) (22, 23, 30, 56). GAE is most prevalent in humans who are immunocompromised (30, 33, 40) and has been reported to occur among individuals infected with the human immunodeficiency virus (HIV) (28). It has been proposed that Acanthamoeba trophozoites access the CNS by passage through the olfactory neuroepithelium (32) or by hematogenous spread from a primary nonneuronal site of infection (23, 24, 33, 53).In immune-competent individuals, GAE is characterized by the formation of granulomas. These cellular aggregates consist of microglia, macrophages, polymorphonuclear cells, T lymphocytes, and B lymphocytes (24, 30). The concerted action of these immune cells results in sequestration of amoebae and is instrumental in slowing the progression of GAE. This outcome is consistent with the observation that granulomas are rarely observed in immunocompromised individuals (34) and in mice with experimentally induced immune suppression following treatment with the cannabinoid delta-9-tetrahydrocannabinol (Δ⁹-THC) (8).Microglia are a resident population of macrophages in the CNS. These cells, along with CNS-invading peripheral macrophages, appear to play a critical early effector role in the control of Acanthamoeba spread during GAE (4, 5, 29, 31). In vitro, microglia have been shown to produce an array of chemokines and cytokines in response to Acanthamoeba (31, 51). However, these factors appear not to have a deleterious effect on these amoebae (29).Acanthamoeba spp. produce serine peptidases, cysteine peptidases, and metallopeptidases (1, 2, 9, 10, 14, 16, 18, 19, 21, 25, 26, 37, 38, 41, 42, 52). In the present study, it is demonstrated that serine peptidases secreted by A. culbertsoni degrade chemokines and cytokines that are produced by immortalized mouse BV-2 microglia-like cells. In addition, soluble factors present in cocultures of A. culbertsoni and BV-2 cells induced apoptosis of the BV-2 cells. Collectively, these results suggest a mode through which A. culbertsoni can evade immune responsiveness in the CNS.     

Lipoprotein Lipase and Hydrofluoric Acid Deactivate Both Bacterial Lipoproteins and Lipoteichoic Acids,but Platelet-Activating Factor-Acetylhydrolase Degrades Only Lipoteichoic Acids

To identify the Toll-like receptor 2 ligand critically involved in infections with gram-positive bacteria, lipoprotein lipase (LPL) or hydrogen peroxide (H₂O₂) is often used to selectively inactivate lipoproteins, and hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) is used to selectively inactivate lipoteichoic acid (LTA). However, the specificities of these chemical reactions are unknown. We investigated the reaction specificities by using two synthetic lipoproteins (Pam₃CSK₄ and FSL-1) and LTAs from pneumococci and staphylococci. Changes in the structures of the two synthetic proteins and the LTAs were monitored by mass spectrometry, and biological activity changes were evaluated by measuring tumor necrosis factor alpha production by mouse macrophage cells (RAW 264.7) following stimulation. PAF-AH inactivated LTA without reducing the biological activities of Pam₃CSK₄ and FSL-1. Mass spectroscopy confirmed that PAF-AH monodeacylated pneumococcal LTA but did not alter the structure of either Pam₃CSK₄ or FSL-1. As expected, HF treatment reduced the biological activity of LTA by more than 80% and degraded LTA. HF treatment not only deacylated Pam₃CSK₄ and FSL-1 but also reduced the activities of the lipoproteins by more than 60%. Treatment with LPL decreased the biological activities by more than 80%. LPL also removed an acyl chain from the LTA and reduced its activity. Our results indicate that treatment with 1% H₂O₂ for 6 h at 37°C inactivates Pam₃CSK₄, FSL-1, and LTA by more than 80%. Although HF, LPL, and H₂O₂ treatments degrade and inactivate both lipopeptides and LTA, PAF-AH selectively inactivated LTA with no effect on the biological and structural properties of the two lipopeptides. Also, the ability of PAF-AH to reduce the inflammatory activities of cell wall extracts from gram-positive bacteria suggests LTA to be essential in inflammatory responses to gram-positive bacteria.Bacterial sepsis is a leading cause of death within intensive care units (43). Although bacterial sepsis was traditionally associated with gram-negative (Gr−) bacteria, recently, the prevalence of sepsis caused by gram-positive (Gr+) bacteria has rapidly increased (2, 3, 38). In fact, in 2000, Gr+ bacteria accounted for 52% of sepsis cases whereas Gr− bacteria accounted for only 37.6% (7, 31, 38). In bacterial sepsis, the innate immune system provides both the initial immune responses and the early inflammatory responses (1, 8, 12). Early responses to infections with Gr+ and Gr− bacteria have been shown in previous studies to involve different cytokine profiles (9, 16, 25, 51, 54). Other studies have found that infections with Gr− bacteria activate Toll-like receptor 4 (TLR4) primarily with lipopolysaccharide (LPS), a membrane component of Gr− bacteria (26, 27, 44, 53). In contrast, infections with Gr+ bacteria involve TLR2, but the nature of the key TLR2 ligand is still controversial (34, 52, 56).Two components of the cell walls of Gr+ bacteria have been proposed to be TLR2 ligands. One group of studies suggests that lipoteichoic acid (LTA) is the key ligand (10, 46, 49, 57). LTA is a polyphosphate attached to the cell membrane via a diacyl glycolipid and is an abundant component of the envelopes of Gr+ bacteria (47). Highly purified LTA, as well as its synthetic analogs, has been shown to trigger TLR2-mediated inflammatory responses (10, 15, 20, 35). However, the biological role of the LTA is unclear because it is difficult to purify natural LTA without introducing contaminants or damaging the structure of the LTA (41). Another group proposes bacterial lipoproteins as the critical ligand (22). Lipoproteins are a functionally diverse class of bacterial membrane proteins characterized by an N-terminal lipid moiety (4) and are TLR2 ligands (22-24). Although synthetic analogs of lipoproteins were found to be potent TLR2 ligands (5, 6, 42), natural lipoproteins are difficult to purify, and their properties are poorly understood.To avoid the technical difficulties involved in purification, a different investigational approach was developed. This approach uses methods to selectively inactivate either LTA or lipoproteins in bacterial culture supernatants or crude bacterial cell wall extracts (22-24, 49). LTA inactivation is usually performed with hydrofluoric acid (HF) or platelet-activating factor-acetylhydrolase (PAF-AH) (23, 48, 49), which, respectively, hydrolyzes the phosphodiester bonds in the LTA or deacylates one of its acyl chains (17, 28, 36, 55). Lipoprotein inactivation is commonly achieved by deacylation with a lipoprotein lipase (LPL) or by oxidation with hydrogen peroxide (H₂O₂) (22, 24, 62). Despite their wide use, the reaction selectivities of these methods have not been evaluated. Thus, we investigated the reaction specificities of these methods by studying the impacts of these four reactions on the biological properties as well as the chemical structures of LTA and lipoprotein analogs.     

Clonal Analysis of Colonizing Group B Streptococcus,Serotype IV,an Emerging Pathogen in the United States

Colonizing group B Streptococcus (GBS) capsular polysaccharide (CPS) type IV isolates were recovered from vaginal and rectal samples obtained from 97 (8.4%) nonpregnant women of 1,160 women enrolled in a U.S. multicenter GBS vaccine study from 2004 to 2008. Since this rate was much higher than the rate of prevalence of 0.4 to 0.6% that we found in previous studies, the isolates were analyzed by using surface protein profile identification, pulsed-field gel electrophoresis (PFGE), and multilocus sequence typing (MLST) to characterize them and identify trends in DNA clonality and divergence. Of the 101 type IV isolates studied, 53 expressed α and group B protective surface (BPS) proteins, 27 expressed BPS only, 20 expressed α only, and 1 had no detectable surface proteins. The isolates spanned three PFGE macrorestriction profile groups, groups 37, 38, and 39, of which group 37 was predominant. The isolates in group 37 expressed the α and BPS proteins, while those in groups 38 and 39 expressed the α protein only, with two exceptions. MLST studies of selective isolates from the four protein profile groups showed that isolates expressing α,BPS or BPS only were of a new sequence type, sequence type 452, while those expressing α only or no proteins were mainly of a new sequence type, sequence type 459. Overall, our study revealed a limited diversity in surface proteins, MLST types, and DNA macrorestriction profiles for type IV GBS. There appeared to be an association between the MLST types and protein expression profiles. The increased prevalence of type IV GBS colonization suggested the possibility that this serotype may emerge as a GBS pathogen.Group B Streptococcus (GBS) (Streptococcus agalactiae) is a leading cause of neonatal infection in the United States, with maternal vaginal or rectal colonization often resulting in the transmission of GBS to the infant during the perinatal period (8, 23). GBS isolates are classified according to nine capsular polysaccharide (CPS) types: types Ia, Ib, and II to VIII and the recently proposed type IX (9, 15, 21, 23, 46, 52). Isolates that do not express any of the known CPS types are designated nontypeable (NT) (2, 6, 21, 40). In addition to CPS, GBS may express one or more surface-localized proteins, including the α and β components of the c protein (24); the alpha-like R proteins, specifically R1, R4(Rib), and R1,R4 (also known as Alp3) (14, 17, 19, 30, 40); and the group B protective surface (BPS) protein (12). Certain protein profiles are associated with each capsular polysaccharide CPS type (2), for example, the c(α only) protein with types Ia and II, c(α + β) with type Ib, and R4(Rib) with type III (2, 14). BPS, expressed by fewer than 3% of colonizing isolates, can be found alone or with another protein in type Ia, II, and V isolates (12, 14).In the United States, the predominant serotypes over the past 2 decades, constituting 70 to 75% of all GBS isolates, have been type Ia, type III, and the more recently emerged type V (14, 15, 20, 52). The remaining isolates consisted primarily of types Ib and II, with types IV, VI, VII, and VIII making up a small fraction of the isolates. We found type IV to represent between 0.4 and 0.6% of colonizing GBS isolates (14, 15), but only rare type IV isolates were found in invasive GBS disease during that same time period (14, 43, 52).In contrast to the previously low percentage of type IV isolates reported for the United States, recent studies in the United Arab Emirates, Turkey, and Zimbabwe showed large proportions of type IV isolates among their GBS isolates. In the United Arab Emirates, type IV was the predominant serotype among colonized pregnant women, representing 26.3% of the GBS isolates (1). In eastern Turkey, it was the second most common serotype, at 8.3%, among colonizing isolates (10), and in Zimbabwe, it was the fourth most common serotype, comprising 5.1% of GBS isolates from colonized pregnant women and 4.0% of all GBS isolates from various sites, including blood and cerebrospinal fluid (CSF), from hospitalized patients (36).Immunization studies of humans (3, 28) and protection studies with mice (37) have shown the potential of vaccines against the common GBS serotypes to prevent invasive neonatal GBS disease through the vaccination of pregnant women (3, 28). The GBS strains described here are from a phase II randomized, double-blinded clinical trial of a GBS serotype III-tetanus toxoid (CPS III-TT) vaccine to prevent the vaginal acquisition of GBS type III in nonpregnant women in three areas of the United States: Pittsburgh (PA), Georgia, and Texas (S. Hillier, unpublished data). Because we found type IV isolates for almost 10% of these patients, we examined the type IV isolates for surface proteins and clonality.Pulsed-field gel electrophoresis (PFGE) was used in this analysis because it is a widely used method that can further characterize GBS isolates within particular CPS type and/or protein profile groups (2, 4, 6, 48). Multilocus sequence typing (MLST) was performed in order to assess the general relatedness of strains within and across laboratories (25, 50). Together, the discriminatory power of PFGE and the objectivity of MLST gave insight into the GBS type IV population genetic structure and the identification of emerging clones (2, 5, 13, 18, 19).     

Glyceraldehyde-3-Phosphate Dehydrogenase Is a Surface-Associated,Fibronectin-Binding Protein of Trichomonas vaginalis

Trichomonas vaginalis colonizes the urogenital tract of humans and causes trichomonosis, the most prevalent nonviral sexually transmitted disease. We have shown an association of T. vaginalis with basement membrane extracellular matrix components, a property which we hypothesize is important for colonization and persistence. In this study, we identify a fibronectin (FN)-binding protein of T. vaginalis. A monoclonal antibody (MAb) from a library of hybridomas that inhibited the binding of T. vaginalis organisms to immobilized FN was identified. The MAb (called ws1) recognized a 39-kDa protein and was used to screen a cDNA expression library of T. vaginalis. A 1,086-bp reactive cDNA clone that encoded a protein of 362 amino acids with identity to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was obtained. The gapdh gene was cloned, and recombinant GAPDH (rGAPDH) was expressed in Escherichia coli cells. Natural GAPDH and rGAPDH bound to immobilized FN and to plasminogen and collagen but not to laminin. MAb ws1 inhibited binding to FN. GAPDH was detected on the surface of trichomonads and was upregulated in synthesis and surface expression by iron. Higher levels of binding to FN were seen for organisms grown in iron-replete medium than for organisms grown in iron-depleted medium. In addition, decreased synthesis of GAPDH by antisense transfection of T. vaginalis gave lower levels of organisms bound to FN and had no adverse effect on growth kinetics. Finally, GAPDH did not associate with immortalized vaginal epithelial cells (VECs), and neither GAPDH nor MAb ws1 inhibited the adherence of trichomonads to VECs. These results indicate that GAPDH is a surface-associated protein of T. vaginalis with alternative functions.Trichomonas vaginalis, an extracellular protozoan parasite, is the cause of trichomonosis, the most prevalent nonviral sexually transmitted disease (47). In women, vaginitis due to T. vaginalis clinically manifests with symptoms of vaginal itching, odor, and discharge. Adverse health outcomes for women with this sexually transmitted disease include cervical cancer (46) and preterm delivery and low-birth-weight infants (25). There is a relationship between seropositivity to T. vaginalis and prostate cancer (43). This disease is significant due to its association with human immunodeficiency virus (33, 45). More recently, persistent, undetected T. vaginalis infections associated with asymptomatic carriage were found among women (40).T. vaginalis penetration of the mucous layer (28), followed by adherence to vaginal epithelial cells (VECs), is preparatory for colonization (9, 10). VEC adherence by parasites is mediated by numerous distinct trichomonad surface adhesins (5, 10, 18). Brief contact of T. vaginalis with VECs and fibronectin (FN) elicited dramatic changes in parasite morphology, suggesting a host-specific signaling of parasites (8, 9). Importantly, iron and cell contact by parasites each upregulated the expression of adhesins in a coordinated fashion via distinct mechanisms (2, 4, 6, 21, 29). Genetic approaches using antisense (AS) inhibition of synthesis (36, 37) and heterologous expression in Tritrichomonas foetus (26, 36) have reaffirmed the role of these T. vaginalis proteins as adhesins. T. vaginalis organisms secrete or release numerous metabolic enzymes, including adhesin AP65 (decarboxylating malic enzyme), α-enolase, and glyceraldehyde-3-phosphate dehydrogenase (GAPDH) during growth and multiplication (27). AP65 and α-enolase were found to reassociate with the parasite surface for the expression of adhesin function (19) and binding to plasminogen (35), respectively.There is an increased awareness of the existence of metabolic enzymes on the surfaces of bacterial pathogens, yeast, and parasites (12, 24, 35). These surface-associated enzymes appear to be novel virulence factors (17, 22, 38, 39). The anchorless glycolytic enzymes GAPDH (13, 31, 38) and α-enolase (39) are present on the surface of group A streptococcus. The surface-associated GAPDH of Candida albicans binds with strong affinity to FN and laminin (22). In enterohemorrhagic Escherichia coli and enteropathogenic E. coli, GAPDH is an extracellular protein that binds human plasminogen and fibrinogen and also interacts with intestinal epithelial cells (17).We demonstrate that GAPDH is another enzyme on the surface of T. vaginalis. A monoclonal antibody (MAb) that inhibited parasite associations with FN was immunoreactive with GAPDH. Importantly, iron was found to regulate gene expression and synthesis and surface placement of GAPDH. Both low-iron-grown trichomonads and AS-transfected parasites with decreased amounts of GAPDH had smaller amounts of surface GAPDH and corresponding lower levels of binding to FN. GAPDH was not involved in adherence of trichomonads to immortalized VECs. Interestingly, as with other microbial pathogens, T. vaginalis GAPDH also bound plasminogen and collagen but not laminin (17, 22).     

Development and Validation of a Multiplex Real-Time PCR Assay for Simultaneous Genotyping and Human T-Lymphotropic Virus Type 1, 2, and 3 Proviral Load Determination

The human T-lymphotropic virus (HTLV) proviral load remains the best surrogate marker for disease progression. Real-time PCR techniques have been developed for detection and quantification of cosmopolitan HTLV type 1a (HTLV-1a) and HTLV-2. Since a growing level of diversity in subtypes and genotypes is observed, we developed a multiplex quantitative PCR for simultaneous detection, genotyping, and quantification of proviral loads of HTLV-1, 2, and 3. Our assay uses tax type-specific primers and dually labeled probes and has a dynamic range of 10⁵ to 10 HTLV copies. One hundred sixty-three samples were analyzed, among which all of the different subtypes within each HTLV genotype could be detected. The performance of proviral load determination of our multiplex assay was compared with that of a previously published HTLV-1 singleplex quantitative PCR based on SYBR green detection, developed at a different institute. Linear regression analysis showed a statistically significant (P < 0.0001) and strong (r² = 0.87) correlation between proviral load values measured with the two distinct real-time PCR assays. In conclusion, our novel assay offers an accurate molecular diagnosis and genotyping, together with the determination of the proviral load of HTLV-infected individuals, in a single amplification reaction. Moreover, our molecular assay could offer an alternative when current available serological assays are insufficient.Since the discovery of human T-lymphotropic virus type 1 (HTLV-1) in 1980 (16, 40), three other genotypes and 10 subtypes have been recognized. The precise geographical distribution and the clinical consequences of these infections are still a matter of debate. This can be attributed at least in part to the fact that there are insufficient accurate tools for HTLV diagnosis, genotyping, and measurement of viral burden.HTLV-1 is endemic in several geographical areas, including sub-Saharan Africa, South America, the Caribbean Islands, Japan, and Melanesia. It has been estimated that worldwide 10 to 25 million people are infected with this retrovirus (41, 53). Most HTLV-1-infected individuals remain asymptomatic throughout their lifetimes. However, 5 to 10% of infected people develop clinical complications, among which adult T-cell leukemia/lymphoma (ATLL) and HTLV-1-associated myelopathy/tropical spastic paraparesis (HAM/TSP) are the most severe. Other manifestations of HTLV-1 infection include infective dermatitis (25), uveitis (34), arthritis (38), and Strongyloides stercoralis infection (53). Some of these manifestations could accelerate disease development and/or progression (12, 16). For HTLV-1, a distinction is made between seven subtypes: the worldwide, cosmopolitan subtype HTLV-1a; the Central African subtypes HTLV-1b, -d, -e, -f, and -g; and the Australo-Melanesic subtype HTLV-1c (8, 23, 41, 52).HTLV-2 was discovered in 1982. This retrovirus is endemic in Amerindian and pygmy populations and epidemic in intravenous drug users (16, 49). In contrast to the case for HTLV-1, convincing epidemiological demonstrations of a definitive etiological role of HTLV-2 in human disease are limited. Nevertheless, HTLV-2 has been linked with the development of neurological disorders similar to HAM/TSP, with arthritis, with pulmonary disorders, and with increased mortality (2, 16, 42). HTLV-2 is divided into three subtypes, namely, HTLV-2a and HTLV-2b, mostly found on the American continent, and HTLV-2d, mostly found in Africa (10, 41, 44, 52).In 2005, two more genotypes, HTLV-3 and -4, were discovered in asymptomatic individuals from Cameroon (6, 7, 47, 56). To date, no diseases have been reported in association with HTLV-3 or -4. Further research is needed to determine the distribution and prevalence as well as the pathogenicity of these two new genotypes.The routine diagnosis of HTLV infections is based on conventional serological techniques such as enzyme-linked immunosorbent assay and Western blotting. However, among samples infected with HTLV-1 or HTLV-2, the proportion of seroindeterminate results is high (20, 21, 28, 57). Moreover, in the cases of HTLV-3 and HTLV-4, an indeterminate Western blot pattern appears to be the rule rather than the exception (6, 29). To confirm and/or support serological assays, diagnostic HTLV PCR techniques were created (51, 54). In the next phase, real-time or quantitative PCR (qPCR) assays were developed that confirm the diagnosis and at the same time quantify the HTLV proviral load (PVL). The majority of the published HTLV qPCR assays are singleplex assays, which detect one HTLV genotype per amplification reaction and hereby were developed for the most prevalent variant of HTLV-1, the cosmopolitan HTLV-1a, or for HTLV-2 infection (11, 22, 26, 32, 55). Multiplex qPCR allows the simultaneous detection and amplification of two or more target DNA sequences in only one amplification reaction. To our knowledge, one specific and one generic biplex qPCR for HTLV-1 and -2 (13, 26) and, just recently, one triplex qPCR for HTLV-1, -2, and -3 have been described (3).To address the current problems with HTLV diagnosis and quantitation, taking into account the diversity in HTLV genotypes and subtypes, we developed a novel triplex qPCR assay for simultaneous detection, genotyping, and quantification of PVL of HTLV-1, -2, and -3 infections. In the future, HTLV-4 can be incorporated into our qPCR technique, provided that viral cell culture is possible. Furthermore, considering the increasing number of HTLV qPCR techniques available at present, together with the lack of validation, we performed the first comparative analysis between two qPCR assays developed at different institutions.     

Molecular Identification and Susceptibility of Trichosporon Species Isolated from Clinical Specimens in Qatar: Isolation of Trichosporon dohaense Taj-Aldeen,Meis & Boekhout sp. nov.

Trichosporon species have been reported as emerging pathogens and usually occur in severely immunocompromised patients. In the present work, 27 clinical isolates of Trichosporon species were recovered from 27 patients. The patients were not immunocompromised, except for one with acute myeloid leukemia. Sequence analysis revealed the isolation of Trichosporon dohaense Taj-Aldeen, Meis & Boekhout sp. nov., with CBS 10761^T as the holotype strain, belonging to the Ovoides clade. In the D1-D2 large-subunit rRNA gene analysis, T. dohaense is a sister species to T. coremiiforme, and in the internal transcribed spacer analysis, the species is basal to the other species of this clade. Molecular identification of the strains yielded 17 T. asahii, 3 T. inkin, 2 T. japonicum, 2 T. faecale, and 3 T. dohaense isolates. The former four species exhibited low MICs for five antifungal azoles but showed high MICs for amphotericin B. T. dohaense demonstrated the lowest amphotericin B MIC (1 mg/liter). For the majority of T. asahii isolates, amphotericin B MICs were high (MIC at which 90% of isolates were inhibited [MIC₉₀], ≥16 mg/liter), and except for fluconazole (MIC₉₀, 8 mg/liter), the azole MICs were low: MIC₉₀s were 0.5 mg/liter for itraconazole, 0.25 mg/liter for voriconazole, 0.25 mg/liter for posaconazole, and 0.125 mg/liter for isavuconazole. The echinocandins, caspofungin and anidulafungin, demonstrated no activity against Trichosporon species.Trichosporon species are yeast-like fungi, widely distributed in nature and commonly isolated from soil and other environmental sources, which have been involved in a variety of opportunistic infections and have been recognized as emerging fungal pathogens in immunocompromised hosts (19, 79, 80). Disseminated Trichosporon infections are potentially life-threatening and are often fatal in neutropenic patients (7, 22). Although uncommon, pathogenic species of this genus have been reported increasingly, mostly in patients with malignant diseases (3, 6, 9, 10, 11, 20, 32, 44, 47, 48, 63, 77), neonates (18, 56, 84), a bone marrow transplant recipient (22), a solid organ transplant recipient (50), and patients with human immunodeficiency virus (34, 35, 46). Trichosporon has also been reported to cause fungemia (5, 9, 25, 29, 30, 33, 53, 62). Members of the genus Trichosporon have occasionally been implicated as nail pathogens (16, 28, 74) and in subcutaneous infections (66). Trichosporon is considered an opportunistic agent, and therefore, recovery of Trichosporon species capable of growing at 37°C, especially from immunocompromised patients, should be regarded as potentially significant. Several reports have addressed the difficulty of identifying Trichosporon to the species level by physiological and biochemical characteristics (2, 64); therefore, molecular methods based on the sequencing of the internal transcribed spacer (ITS) have been developed (15, 69, 71, 72).In the present paper, we report the isolation of Trichosporon species from clinical specimens over a 4-year period in Qatar, the poor performance of biochemical identification methods, the significance of molecular identification, and the antifungal susceptibility data for the isolates. While investigating the molecular identification of Trichosporon species, we found three strains that do not match any of the published strains in the literature. We describe this organism as Trichosporon dohaense Taj-Aldeen, Meis & Boekhout, sp. nov., the name proposed for this species.     

Immune Defenses against Batrachochytrium dendrobatidis,a Fungus Linked to Global Amphibian Declines,in the South African Clawed Frog,Xenopus laevis

Batrachochytrium dendrobatidis is a chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians. It is regarded as an emerging infectious disease affecting diverse amphibian populations in many parts of the world. Because there are few model amphibian species for immunological studies, little is known about immune defenses against B. dendrobatidis. We show here that the South African clawed frog, Xenopus laevis, is a suitable model for investigating immunity to this pathogen. After an experimental exposure, a mild infection developed over 20 to 30 days and declined by 45 days postexposure. Either purified antimicrobial peptides or mixtures of peptides in the skin mucus inhibited B. dendrobatidis growth in vitro. Skin peptide secretion was maximally induced by injection of norepinephrine, and this treatment resulted in sustained skin peptide depletion and increased susceptibility to infection. Sublethal X-irradiation of frogs decreased leukocyte numbers in the spleen and resulted in greater susceptibility to infection. Immunization against B. dendrobatidis induced elevated pathogen-specific IgM and IgY serum antibodies. Mucus secretions from X. laevis previously exposed to B. dendrobatidis contained significant amounts of IgM, IgY, and IgX antibodies that bind to B. dendrobatidis. These data strongly suggest that both innate and adaptive immune defenses are involved in the resistance of X. laevis to lethal B. dendrobatidis infections.Batrachochytrium dendrobatidis is a newly described chytrid fungus that causes the lethal skin disease chytridiomycosis in amphibians (29). Growing evidence links amphibian declines in Australia, Central America, the western United States, Europe, and Africa to this emerging infectious disease (4, 9, 12, 26, 29, 34-36, 45, 65). B. dendrobatidis colonizes skin cells of adults and the keratinized mouth parts of tadpoles (3, 4, 29, 34) but does not invade other tissues. It is spread by waterborne zoospores that attach to the skin and migrate to the basal layer of the epidermis (3). The pathogen replicates within the epidermal cells and moves to the surface as the cells mature. Emerging zoospores may infect the same host or another nearby host (3, 4, 29, 34). Recent evidence supports the hypothesis that death results from impaired retention of essential ions by the skin resulting in eventual cardiac arrest (63, 64). Some species of amphibians are very resistant to lethal infections of B. dendrobatidis, whereas others are more susceptible (4, 26, 27, 38, 66-68), and the factors that determine resistance or susceptibility are not well understood. Although much is known about amphibian immunity in general (9, 14, 41), there is limited information about specific immune responses against B. dendrobatidis.We hypothesized that resistant species have antimicrobial peptides or antibodies in the mucus that limit initial infections by B. dendrobatidis zoospores and prevent the further colonization of the same host by zoospores emerging from the skin. Previous work has shown that individual purified antimicrobial peptides (11, 44-50, 52, 68) and enriched skin peptides (48, 52, 66-68) from many species can inhibit the growth of B. dendrobatidis zoospores and mature sporangia in vitro. The skin of amphibians is also protected by the adaptive immune system. Antigens in the skin can be transported to the spleen, where an immune response involving both T cells and B cells can occur (9, 14, 41). In mammals and in fish, antibodies are present in mucosal secretions (10, 28, 31, 53), but there have been no previous studies of antibodies in amphibian mucus.X. laevis was chosen as the species to investigate immunity to B. dendrobatidis because this species has been widely used as a model for studies of amphibian immunity since the 1960s (9, 14, 41). X. laevis is quite resistant to the lethal effects of infection with B. dendrobatidis in nature. Infections were detected in archived specimens of X. laevis as early as 1938, and the incidence of infected individuals appears to be constant (∼3%) over the last 60 years (1941 to 2001) (65).We show here that after exposure to B. dendrobatidis, immunocompetent frogs developed a mild infection that is almost completely cleared by 45 days. Antimicrobial skin peptides inhibited B. dendrobatidis growth, were present at effective concentrations in resting frogs, and increased in number when frogs were exposed to an “alarm” stress. Treatment with norepinephrine depleted skin peptide stores and increased host susceptibility to infection. X-irradiation depleted leukocytes in the spleen without altering the capacity to secrete skin peptides, and the infection intensity was significantly greater in the irradiated frogs. Immunization with heat-killed B. dendrobatidis induced significantly elevated pathogen-specific IgM and IgY in the serum detectable for at least 1 month after the last immunization. In addition to antimicrobial peptides, skin mucus samples from frogs exposed to B. dendrobatidis 5 months earlier contained antibodies of all three immunoglobulin classes that bind B. dendrobatidis. Whether the mucosal antibodies are protective will be determined in ongoing studies. Collectively, these data demonstrate that X. laevis is a good model species to study immune defenses against B. dendrobatidis, and both innate and adaptive immune mechanisms appear to be involved in the resistance to lethal infections.