However, among non-HAART users-ever, the oldest age group had the highest incidence rate ratio compared to younger individuals ). While the incidence of KS decreased with age, older HIV-infected persons who do not receive HAART are still at increased risk of KS. As KS remains an important malignancy among HIV-infected persons, earlier HIV diagnoses and HAART initiation, particularly in older HIV-infected persons is warranted.The life expectancy for HIV-positive individuals has improved over time due to increasing access to highly active antiretroviral therapy (HAART). Yet, as the HIV-positive population ages, their risk of developing cancers also increases. Studies of Kaposi's sarcoma (KS) among elderly HIV-infected persons are quite limited. We examined the age patterns of KS incidence and an association between age and KS risk in a US cohort of 3458 HIV-infected men, the Multicenter AIDS Cohort Study (MACS). Poisson distribution was used to calculate incidence rates and respective 95% confidence intervals (95% CIs). Cox proportional hazards regression was performed to examine the association between age and KS risk. There were 534 incident KS cases with a total follow-up time of 25,134 person-years. The overall KS incidence rate was 2.13 per 100 person-years (95% CI: 1.95–2.32) . Overall, KS frequency and incidence declined with age, even in the oldest age group ( Kaposi's sarcoma (KS) is caused by human herpesvirus-8 (HHV-8), which was first discovered in 1984 by Chang et al. ).AIDS patients have an increased risk of certain types of cancer due to their immunocompromised status –6. As HIIn HIV-infected persons, KS is still one of the most common cancers. KS can have profound effects on quality of life and may become a debilitating condition, especially in the context of the other comorbidities associated with AIDS. An HIV-infected person is 3640 times more likely to have KS than an HIV-uninfected person . AlthougStudies of KS among older HIV-infected persons and AIDS patients are limited. Recent reports indicate that cancer incidence has risen rapidly among this aging population , 15. It Data used for the current study were obtained from the public dataset (release 18) of the Multicenter AIDS Cohort Study (MACS), an ongoing cohort study of HIV-infected and uninfected men who have sex with men (MSM) in the US. MACS is described in detail elsewhere . BrieflyThe MACS study protocol includes a baseline visit and follow-up visits at 6-month intervals. During both baseline and follow-up visits, study participants were administered a standardized questionnaire by trained interviewers. Information obtained through the interview included: sociodemographic and behavioral characteristics , health service utilization, medical history, medication use , and HIV-related symptoms , 18. Alsn = 3334). Thus, the remaining sample of 3458 men who were either HIV-seropositive at baseline or seroconverted during the cohort was used for the current analysis.MACS has recruited 6792 HIV-infected and HIV-uninfected MSM. For the current analyses, participants were excluded if they remained HIV-uninfected for the duration of follow-up . Race/ethnicity was categorized into three groups . Employment status was dichotomized into current employment and no current employment. Education level was grouped into three groups . Individual gross income was categorized into four groups . Current smoking status was defined as “Yes” or “No”.HAART use was defined as a binary variable , according to the DHHS/Kaiser Panel . AccordiIncident cases of KS among HIV-seropositive men were defined as persons who did not have KS at the baseline visit but developed KS during the follow-up period. Incident cases of KS among HIV-seroconverters were defined as persons who did not have KS at the seroconversion visit but developed KS during the follow-up period. The overall time to follow-up was from 1984 (starting at first enrollment) to 2005 (the cutoff period for the provided public dataset). Among HIV-seropositive men, follow-up time was defined for each subject as the time from the date of enrollment into the cohort to the date of diagnosis for KS cases, or to the date of last recorded visit for subjects who did not develop KS. For HIV-seroconverters, follow-up time was from the date of seroconversion to the date of KS diagnosis for KS cases, or from the date of seroconversion to the date of last recorded visit for KS-free participants. Age at diagnosis or last visit was calculated for KS cases or KS-free participants, respectively. It was then, categorized into three groups as mentioned above.P < 0.0001), we included only CD4 + cell count into the adjusted models. Covariates included in the models were current employment, education level, individual gross income, current smoking status, CD4 + cell count (as a time-dependent variable), and enrollment period. Enrollment period was included in the adjusted models because a previous analysis using MACS data showed that there was a difference in HAART adherence between Caucasians and African Americans between the two enrollment periods  Table Ptrend < 0.0001). Those who did not receive HAART were significantly more likely to develop KS than those who did receive HAART in each of the age groups <40, 40–49, and ≥50 . The protective effect of HAART was also observed in all three race/ethnicity groups. A similar observation was found when stratifying by HIV-seropositive only versus HIV-seroconverters only  is similar to that found in the HIV/AIDS Cancer Match Study (HACM) that examined Caucasian MSM from six states and five metropolitan areas during the 1981–1996 period (incidence rate: 2.4 per 100 person-years) . Given tThe declining trend of KS incidence with age found in our analysis is also consistent with findings from other studies , 28, 29.Ptrend < 0.0001). Also, the IRR of KS was the highest in the oldest age groups for both HIV-infected men and HIV-seroconverters. Although the incidence of KS is decreasing, it is important to note that KS and non-Hodgkin lymphoma are still the most common cancers in the US AIDS population [Another important point from our analysis is that although there is a declining trend of KS incidence with age, the risk of KS development is higher in every age group among those without HAART compared to those with HAART (pulation  despite pulation . Furtherpulation . Specifipulation  reportedpulation , 38 repopulation –41, partWe also found that the incidence of KS decreased in both older age groups; however, in multivariable analyses, the risk of KS in the oldest group was higher than that in 40- to 49-year-olds. Further studies are, therefore, warranted to confirm the KS incidence patterns among the older population with or without HAART usage.The main strength of our analysis was the use of the largest cohort study among HIV-infected men in the US to examine the age patterns of KS incidence. This well-designed cohort allowed us to calculate accurate estimates of KS incidence with age. The design of this cohort also makes it possible to identify the date of seroconversion and to calculate follow-up time accurately. Also, too our knowledge, current analysis is one of the only studies to examine KS in HIV-seroconverters. One limitation of this analysis is that because the cohort only included MSM and most of them were Caucasian American, our results may not be generalizable to HIV-infected women or to mixed race/ethnic populations. Another limitation is that since the data provided were followed to 2005, the definition of HAART used in our study might not be comparable to the new definition in 2011, particularly because several new HIV medications have been approved since 2005. The other limitation is that information on KSHV serostatus was not available in the current provided dataset.In summary, we found declining incidence rates of KS with age among HIV-infected men in the largest US cohort. Furthermore, we reported that HAART has great impact in decreasing the overall KS incidence in this cohort. Among the HIV-infected older population, the KS risk is higher in those without HAART compared to those with HAART. Our findings are consistent with results from previous studies and provide information on the directions of age and period effects on KS risk. Our results have substantial clinical and public health implications related to the low usage of HAART in HIV/AIDS-affected populations, especially in older populations. As KS remains an important malignancy among HIV-infected persons, earlier HIV diagnosis and HAART initiation, particularly in older HIV-infected persons should remain a public health priority.


Human T-lymphotropic virus-1 (HTLV-1) can cause adult T-cell leukemia/ lymphoma (ATL/L), which is a rare and aggressive type of blood cancer. Herein, we report a case of ATL/L in a middle-aged man with unusual jaw presentations. The patient presented with mandibular, maxillary and palatal bony hard expansion, accompanied by generalized tooth mobility six months prior to admission to the Department of Oral Medicine. The panoramic radiograph showed generalized rarefaction of jaw bones. After laboratory examinations and bone marrow aspiration, ATL/L was diagnosed in association with HTLV-1. The patient underwent chemotherapy.Although the majority of infections associated with HTLV-1 are asymptomatic, some patients may develop blood diseases such as ATL/L and neurological disorders, mainly HTLV-1 associated myelopathy and tropical spastic paraparesis. ATL/L is a rare hematological malignancy in oral cavity that should be included in the differential diagnosis of cases with jaw swelling or generalized demineralization. Serum levels of anti-HTLV-1 antibodies should be examined in suspicious patients, particularly in endemic regions. Human T-cell lymphotropic virus HTLV-1) is a retrovirus, infecting CD4+ lymphocytes. In Iran, the main highly endemic regions for HTLV-1 are the Northeastern regions, particularly Mashhad and Neyshabur.[ is a retProper diagnosis of ATL/L would inevitably lead to appropriate treatment and better prognosis. To the best of our knowledge, concurrent expansion of mandibular and maxillary bones has not been reported thus far; however, some cases of ATL/L accompanied by facial bone involvement have been reported.-3In this paper, we report a case of ATL/L with unusual manifestations including mandibular, maxillary, and palatal expansion as well as tooth mobility in a patient with HTLV-1.The patient was a 45-year-old man who had referred to Mashhad Dental School for dental extraction. The patient had complaints of hypermobility in some teeth over the last four months. Six months earlier, he had noticed the enlargement of the jaw concurrent with pain in the muscles and bones of feet and hands after being hospitalized for appendectomy. Furthermore, the patient had a prior history of hospitalization due to fever, vomiting, nausea, and diarrhea; a diagnosis of acute urinary infection had been established.On palpation, the swelling was non-tender, bony hard, and non-compressible with the expansion of both buccal and lingual cortical plates extending from the right second molar to the left second molar in mandibular and maxillary bones as well as hard palatal bones . The patient’s teeth were all loose and displaced. Diastema, which was observed in all teeth in both the upper and lower dental arches, appeared after jaw expansion. According to the patient's self-report, the oral mucosa was normal. We did not find any palpable lymphadenopathy in the head or neck region. Radiographic and laboratory tests were performed. The panoramic radiograph revealed the generalized rarefaction of jaw bones; the inferior alveolar canal was unclear . ThinninIn the midline, also the borders of maxillary sinus and hard palate were ill-defined . OcclusaDifferent laboratory tests were requested which showed a mild increase in white blood cell (WBC) count and an increase in alkaline phosphate (ALP) . AlthougAfter about 50 days, the patient was referred to our department again. Another blood test was ordered. As the comparison between the results of the two tests indicated, WBC count increased from 15,000/μL to 53,900/μL and ALP elevated from 500U/L to 1530 U/L. The rapid increase in ALP could be an indicator of osteolysis and increased WBC count revealed the malignant proliferation of bone marrow cells.The differential diagnosis included different malignancies, especially hematologic disorders such as leukemia/lymphoma. The patient was referred to a hematologist and an oncologist for further evaluation. Anti-HTLV-1 antibodies were detected by means of enzyme-linked immunosorbent assay (ELISA) and Western blot analysis. Bone marrow aspiration showed a hypercellular bone marrow and pleomorphic lymphocytes with large flower-shaped nuclei; a high percentage of cells were atypical. A diagnosis of ATL/L was established. It seems that the first signs of this malignancy were observed in the jaw bones. The patient underwent cyclophosphamide, doxorubicin, vincristine, and prednisone (CHOP) chemotherapy.Three years after the completion of chemotherapy, cutaneous lesions presented as maculopapular eruptions. Although the patient’s cancer was in the remission phase, he was receiving some medications. Histopathological evaluation after the incisional biopsy of cutaneous lesions showed non-specific dermatitis. According to the dermatologist’s outlook, the cutaneous lesions could be related to HTLV-1 or drug-induced dermatitis. During the patient’s visit, complete blood cell count did not show any abnormalities. HTLV-1 infection is endemic in some geographical regions and people residing in these areas may be infected by HTLV-1, without showing any clinical symptom. In these endemic regions, patients with leukemia/lymphoma should be examined for serum anti-HTLV-1 antibody level. Additionally, polymerase chain reaction (PCR) could be applied to detect the HTLV-1 genome in paraffin-embedded tissues. A previous community-based seroepidemiologic study demonstrated that the overall prevalence of HTLV-1 infection in the whole population of Mashhad city was 2.12%.[Generally, most individuals infected with HTLV-1 are asymptomatic carriers; however, a few patients would present some conditions such as ATL/L or tropical spastic paraparesis which is a chronic progressive myelopathy. ATL/L isThe mechanism involved in the malignant behavior of infected T-cells is still unknown. Disturbances in the genetic structure of host cells are proposed as risk factors for the development of oncogenic events. ATL/L isOur patient had hypercalcemia, positive serological tests for HTLV-1, and a total WBC count of 50,000/μL which are considered as indicators of ATL/L. Although other cases of ATL/L in HTLV-1-positive individuals have been reported in the literature, a few cases were initially diagnosed based on their oral manifestations., 9In a previous study, one case of ATL/L associated with HTLV-1 was reported in which the first clinical finding was swelling of the cheek. Although dental infection was suspected, further evaluations showed that the lesion extended from the maxillary alveolus to maxillary sinus. Also, a In Brazil, one case of ATL/L was reported in a 30-year-old woman, presenting with a painful ulcerated lesion in the mucosa covering the hard palate. Furthermore, involvement of several bones, lungs, and axillary lymph nodes was noted. However,Furthermore, some cases of ATL/L have been reported in other parts of the body except the head and neck in non-endemic regions such as Lebanon. Two cases of ATL/L accompanied by lymphocytosis and severe hypercalcemia were reported in this region. MoreoverOur patient did not show any remarkable symptoms or signs exemplifying ATL/L during his first visit, similar to the mentioned cases. In our case, we considered leukemia/lymphoma in the differential diagnosis, given the generalized expansion of mandibular and maxillary bones, diffuse rarefaction of jaw, and tooth mobility. In fact, in the oral cavity, leukemia and lymphoma can cause the expansion of jaw bones, tooth mobility, and multiple ill-defined non-corticated radiolucent lesions. Additionally, non-Hodgkins lymphoma can lead to an increase in lymphocytic lactate dehydrogenase and ALP which is an indication of bone loss. A severe increase in ALP was found in the present case. Considering the high level of ALP, hypercalcemia, and generalized rarefaction in the patient’s jaw, hyperparathyroidism was included in the differential diagnosis. However, in hyperparathyroidism, other bones are usually involved and patients usually show other signs and symptoms of the disease.Despite the high level of ALP and generalized expansion of mandibular and maxillary bones, Paget’s disease was excluded from the diagnosis since other bones of the patient were not involved. Furthermore, if Paget’s disease only affects the maxilla or mandible, ALP remains within the normal range while inIroi reported a case of a patient who was positive for HTLV-1 with low CD4/CD8 ratio, indicating the presence of cellular immunodeficiency. ThereforOn the other hand, HTLV-1 can induce the formation of maculopapular lesions on the skin. In our patient, maculopapular lesions were observed after chemotherapy was completed. These lesions could be related to HTLV-1 complications or side-effects of chemotherapy; although, histopathologic examination of cutaneous eruption showed non-specific lesions. Regardless of clinical presentations, the prognosis of ATL/L is quite poor. As previATL/L is a rare hematological disorder in the oral cavity. However, physicians and dentists should consider this malignancy in the differential diagnosis of jaw swelling or generalized rarefaction, particularly in HTLV-1 highly endemic regions.


The design of chemical libraries, an early step in agrochemical discovery programs, is frequently addressed by means of qualitative physicochemical and/or topological rule-based methods. The aim of this study is to develop quantitative estimates of herbicide- (QEH), insecticide- (QEI), fungicide- (QEF), and, finally, pesticide-likeness (QEP).In the assessment of these definitions, we relied on the concept of desirability functions.We found a simple function, shared by the three classes of pesticides, parameterized particularly, for six, easy to compute, independent and interpretable, molecular properties: molecular weight, logP, number of hydrogen bond acceptors, number of hydrogen bond donors, number of rotatable bounds and number of aromatic rings. Subsequently, we describe the scoring of each pesticide class by the corresponding quantitative estimate. In a comparative study, we assessed the performance of the scoring functions using extensive datasets of patented pesticides.The hereby-established quantitative assessment has the ability to rank compounds whether they fail well-established pesticide-likeness rules or not, and offer an efficient way to prioritize (class-specific) pesticides. These findings are valuable for the efficient estimation of pesticide-likeness of vast chemical libraries in the field of agrochemical discovery.The online version of this article (doi:10.1186/s13321-014-0042-6) contains supplementary material, which is available to authorized users. Ora. Orain sibraries . Hence, ibraries -7]..in silicIn drug discovery, Lipinski's rule of five (Ro5) is considered to be the reference in defining physicochemical and structural properties profiles for optimal bioavailability of drug candidates . Upper lIn the field of agrochemical discovery, Lipinski's Ro5 approach was quickly adopted to profile agrochemicals, i.e., herbicides, insecticides and fungicides ,12]. In. In12]. To overcome the hard boundaries established by traditional filters for drug-likeness, Bickerton et al.  developeWe consider that the field of agrochemical discovery would benefit from a similar treatment of pesticide-likeness. Thus, in this study, we aim to establish quantitative estimates of pesticide-likeness. Three main classes of pesticides are considered herein, i.e., herbicides, insecticides and fungicides, and, accordingly, we describe the quantitative estimate of herbicide-likeness (QEH), of insecticide-likeness (QEI) and of fungicide-likeness (QEF). We found a simple type of function that accurately describes six physicochemical properties over the three pesticide classes. Furthermore, we compare the performance of this quantitative approach to well known rule-based methods defining pesticide-likeness using a large library of patented compounds for agrochemical applications and discuss the results. For practical reasons and for the purpose of this paper, we will denominate the ensemble of scoring functions dedicated to pesticide-likeness as QEPest-SFs.We applied the concept of desirability  to proviMarketed pesticide set section in Methods). The resulted distributions of the three pesticide-classes were fitted as described in Curve fitting section in Methods. We found six independent  that would accurately fit distributions resulted from molecular properties describing herbicides, insecticides and fungicides. Firstly, we computed a number of 15 molecular descriptors  were joined accordingly for each pesticide-class by computing geometric means. This can be expressed by logarithmic identities, as the exponent of the arithmetic mean of the logarithm transformed dfs (see eq. i) zero to one range, (ii) output values will increase as the balance of the properties becomes more favorable, (iii) if any dfi = 0 (is unacceptable) the geometric mean is null, i.e., if a property is unacceptable the compounds becomes unacceptable.The individual  see eq. . As arguWe denominate the resulted scoring functions as quantitative estimates of herbicide-likeness (QEH), insecticide-likeness (QEI) and fungicide-likeness (QEF), according to the pesticide class. These functions reflect the probability of a molecule to exhibit desirable characteristics as a pesticide. Thereby, we obtained an intuitive quantitative indicator of the likeness of a molecule to match the physicochemical profile of pesticides.In order to model specific properties of large data sets, predictive models often use many descriptors limiting the applicability domains of the model. The more descriptors are used, the greater is the likelihood that a candidate molecule will fall outside the limits of one or more of these descriptors . In our The three main classes of pesticides are: herbicides (against weeds), insecticides (against harmful insect pests), and fungicides (against harmful diseases) ,14],,[In Figure dfs agree with previous findings, and slight variations in the distributions might be reasoned by the various datasets employed.The recent analysis, conducted by Hao et al. , concernGVKBio agrochemical patents collection (AgroSAR) comprises ~ 59 k (58915) unique structures and ~ 413 k (413103) SAR end-points measured in ~110 k (109733) assays. A percentage of 38.7% of the data has been published in the seventies, 29.6% in the eighties and 28.67% in the nineties up to 2005. AgroSAR gathers herbicides, insecticides, fungicides, acaricides, nematocides, bactericides, algaecide, plant growth, biocides, microbiocides and rodenticides in a relational database, manually curated and annotated, easy to query and subset. This database comprises large amounts of unexplored patent data, which can help to improve the discovery of agrochemicals. To our knowledge, this is the only SAR patent database built specifically from patent specifications filed in the agro sector.We selected a subset of potent herbicides, insecticides and fungicides available in AgroSAR, as defined by more than 50% activity obtained at concentrations of 4.5 lb/acre 0.826 kg/ha) for herbicides, 125 ppm for insecticides and 100 mg/L for fungicides (cutoffs established by the medians of the activity data available per class). Hence, after removing marketed pesticides, we retrieved 1105 herbicides, 8983 insecticides and 9371 fungicides , QEI scores ≥0.57 (insecticides), QEF score ≥0.6 (fungicides), QEPmax ≥0.7 and QEPavg ≥0.6 (pesticides). These results, further supported by the cutoff values corresponding to 25% and 75% of the datasets  were assembled for each pesticide class. Using the same six molecular properties, we computed QEH, QEI, QEF, QEPmax and QEPavg also for the decoys sets .Simple rule-based methods that define pesticide-likeness are applied in the early stages of pesticide-discovery programs. Due to their simplicity, these methods serve to trim large chemical libraries to smaller sets, which are supplied to more computational-expensive approaches. In this sense, a challenging exercise for QEPest-SFs would be to recognize pesticides from a larger set of decoys. In consequence, ten times larger sets of randomly chosen representatives from PubChem Compounds (Performance measure section in Methods) plots describing the capacity of QEH, QEI, QEF, QEPmax and QEPavg to recognize the corresponding pesticide sets. A barely increased early enrichment can be seen in the case of QEI retrieving insecticides and, in contrast, QEH retrieved more lately herbicides. The discriminative performance was numerically assessed by AUC (area under the ROC [Performance measure section in Methods) values as reported in Additional file In Figure  the ROC  – see PeQEPest-SFs have the ability to rank compounds whether they fail pesticide-likeness rules or not. In consequence, different cutoffs for the scoring functions provide various levels of sensitivity and specificity. One might be tempted to find optimal cutoffs values for these scoring functions. The results of such an approach are reported in Additional file In this study, we have demonstrated that QEPest-SFs are able to rank compounds according to their herbicide-, insecticides-, fungicide- or pesticide-likeness. These scoring functions are based upon six simple molecular descriptors and a single type of function, parameterized accordingly to provide desirability scores. These quantitative assessments provide increased flexibility compared to traditional rule-based methods. For example, large chemical libraries can be reduced to desirable sizes, profiling pesticide-like molecules at various levels. In the usual pipeline of a drug and agrochemical discovery programs the resulted sets are supplied to more accurate virtual screening methods to increase cost-effectiveness in further experimental steps. For this purpose, we provide a simple Java-based program ("QEPest.jar") to compute QEH, QEI and QEF . The marketed pesticide set was used to derive quantitative estimate scoring functions for herbicide-likeness (QEH), insecticide-likeness (QEI), fungicide-likeness (QEF) and overall pesticide-likeness (QEP).A set of 1685 pesticides  was assembled from The Pesticide Manual  and Comphttp://www.chemaxon.com). Six descriptors, i.e., molecular weight (MW), molecular hydrophobicity , number of hydrogen bond acceptors (HBA), number of hydrogen bond donors (HBD), rotatable bonds (RB), aromatic rings (arR) were used to derive desirability functions for QEPest-SFs. Other hydrophobicity estimation metrics such as MLogP [http://www.talete.mi.it) and BioByte , respectively, and were used accordingly, as required by rule-based methods  [For the assessment of the desirability functions we computed the frequency counts for each class of pesticides, according to the descriptor type-values, i.e., for continuous values (MW and LogP) the optimum bin size was computed with 21 2013) , and for21 2013) .Online Curve Fitting and Surface Fitting Web Site . Depending on the data to be modeled, up to 573 non-linearly, and 23 linearly equations, were fitted.The frequency counts and bins computed for each molecular descriptor served as input for curve fitting processed by means of ZunZun.com versus the false positive rate (FPR = 1- specificity) according to the ranked list. AUC values indicate the ability of a scoring method  to discriminate between two classes of elements, e.g., actives and inactives, and is defined by the area under the ROC. Values range from 0 to 1 (perfect separation), 0.5 suggesting a random spread of the representatives of the two classes.The discriminative power of QEPest-SFs was assessed graphically and numerically by means of receiver operating curve (ROC)  and the SM initiated and supervised the project. SA carried out the calculations, implemented and tested the scoring functions, developed the Java-based program and prepared the manuscript. SFT and AB contributed to data preparation for model development and validation and drafted the manuscript. SRC and AKM provided the AgroSAR patent database and corresponding annotations. All authors read and approved the final manuscript.Supporting Tables and Figures. This pdf file contains nine tables (Table S1's9) and three figures  offering supporting data as referenced throughout the paper. (PDF 396 KB)Additional file 1: Additional file 2: QEPest Java program. In the archive QEPest.zip we provide a simple Java-based program ("QEPest.jar") to compute QEH, QEI and QEF, based on pre-generated descriptors, accompanied by a input example ("data.txt"), an output file ("data.txt.out") and a "readme.txt" file for instructions. (ZIP 7 KB)Authors’ original file for figure 1Authors’ original file for figure 2Authors’ original file for figure 3Authors’ original file for figure 4Authors’ original file for figure 5Authors’ original file for figure 6Authors’ original file for figure 7Authors’ original file for figure 8Below are the links to the authors’ original submitted files for images.


A 6-year-old boy, with no past medical history, presented with progressive headache, vomiting, right side weakness since 3 months without fever or seizures. On examination, he was conscious with a stiff neck and right hemiplegia. Brain computed tomography-scan showed a heterogeneous calcified large mass-like lesion in the left fronto-temporo-parietal region, irregular contrast enhancement and extensive perilesional edema with important mass effect (A-C). There was a strong suspicion of malignant tumor. Routine blood tests and chest X-ray were normal. Magnetic resonance imaging was requested but not made because of the rapid deterioration of the state of consciousness of the child. The patient was operated urgently. At operation there was a large intra-axial yellowish, firm, and relatively avascular lesion. Total excision of the mass was done (D). Histological features were consistent with tuberculoma. Serological test for HIV was negative. The patient improved progressively and was discharged on antituberculous treatment with a good outcome. Intracranial tuberculoma should always be considered in the differential diagnosis of solitary and large focal brain lesions, particularly in patients of tuberculosis endemic areas. In our patient, surgical excision not only helped to establish the histological diagnosis but also helped to resolve the compressive symptoms.


In all European countries, hospital-acquired infections caused by Gram-negative multidrug-resistant microorganisms (GN-MDRO) are a major health threat, as these pathogens cannot be adequately treated anymore, or the start of effective antibiotic treatment is delayed. The efforts to limit the selection and spread of GN-MDRO remains a problem in cross-border healthcare, as the national guidelines on hygiene standards applicable for patients colonized or infected with GN-MDRO in hospitals are not harmonized between European countries.In order to point out the similarities and differences in the national guidelines of Germany and The Netherlands regarding GN-MDRO, guidelines were compared and an expert workshop was organized by the INTERREG IVa project EurSafety Health-net.Enterobacteriaceae requires testing for certain mechanisms causing antibiotic resistance, whereas the German guideline makes use of a newly created classification scheme, based on phenotypic characterization. Besides diagnostic issues, the main difference between the Dutch and German guideline is the divergent evaluation of ESBL-producing Enterobacteriaceae. Special hygiene measures are required for all patients with ESBL-producing Enterobacteriaceae in The Netherlands, whereas the German guideline recommends special precautions only for those cases in which patients are colonized or infected with strains showing co-resistance to ciprofloxacin (“3MRGN”).Both guidelines divide the Gram-negative organisms into subgroups based on bacterial species and antibiotic susceptibility patterns in order to define multidrug-resistant variants of these bacteria. However, the Dutch guideline defines that GN-MDRO The usage of consistent terminology and harmonized diagnostic procedures would improve the possibilities for infection prevention, treatment and patient safety. Prevention of severe non-treatable infections and outbreaks due to MDRO, caused by an increased population seeking medical treatment abroad together with an increased number of highly susceptible individuals demands gathering of regional data, and data comparable between the two sides of the Dutch-German border. The necessity to cooperate multidisciplinary and across borders is required to prevent a post-antibiotic era – in which common infections and minor injuries may lead to death. Enterobacteriaceae and multidrug-resistant Pseudomonas aeruginosa and Acinetobacter baumannii, cause an incremental part of hospital-acquired infections in all European countries [Gram-negative multidrug-resistant microorganisms (GN-MDRO), such as Extended Spectrum β-Lactamase (ESBL) producing or Carbapenem-resistant ountries ,2. This ountries ,4. Besidountries .In Europe, it is estimated that about 25,000 people die as a direct result of GN-MRDO infections every year . Hence, As international travel and international patient care are considered to represent major risk factors for acquiring GN-MDRO, European countries need to implement measures to prevent transmission of GN-MDRO between healthcare institutions abroad and national facilities ,8. ExempEnterobacteriaceae.As these guidelines are not harmonized between countries, definitions, nomenclature of GN-MDRO and the respective precaution measures recommended differ to some respect. For the two countries, which share a common border for 567 km, these differences may lead to special challenges in cross-border patient care. This occurs when patients are treated in hospitals on both sides of the border or are transferred from one country to the other. One of the main goals of this cross-border region is to prevent the spread of especially carbapenemase producing This issue is of special importance, because the Directive 2011/24/EU published by the European Union that came into force on October 25th 2013, clearly outlines the patients’ right to seek healthcare service across European borders. In addition, it has already been demonstrated that patient transfers between hospitals significantly increase the transmission of hospital-acquired infections at the regional and national scale [Therefore, in this article, we describe and compare the current Dutch and German GN-MDRO definitions and prevention guidelines for hospitals. The aim of this article is to point out differences between the two national guidelines and the occurrence of MDRO with regard to the epidemiology, prevention measures and diagnostics that need to be considered when cross-border patient care is performed in order to forestall misunderstandings, ensure patient safety and guarantee sustainable healthcare.www.eursafety.eu) in order to discuss the differences observed between the two national guidelines. About 75 experts in the field of Medical Microbiology and Infection Prevention from Germany and The Netherlands attended the workshop “ESBL/Multidrug Resistant Gram-negatives (MRGN) harmonization of the new guidelines in Germany and The Netherlands” in Nijmegen, The Netherlands, on December 11th, 2013. Stakeholders coming from the cross-border region of Germany and the Netherlands, were able to define their own questions regarding GN-MDRO in the border area. Due to the experts’ different areas of expertise, three dicussion groups were formed. Discussing on the Epidemiology, Infection Prevention or Diagnostics & Treatment of GN-MDRO, the following questions were tried to answer:i)What are the similarities and differences between the Dutch and German Guideline?ii)May problems be caused by cross-border patient care?iii)Which actions are required to harmonize both guidelines?Primarily, the current versions of the German and Dutch guidelines on GN-MDRO prevention in healthcare facilities were analysed with respect to similarities and differences taking into account definitions, nomenclatures and hygienic precaution measures to be taken by healthcare workers ,10. Aftesensu stricto). GN-MDRO are designated “BRMO” (Bijzonder resistente micro-organismen) according to the Dutch guideline and “3MRGN” (Multi-resistente Gramnegative Erreger), e.g. ESBL-producing K. pneumoniae with resistance to ciprofloxacin, or “4MRGN”, e.g. resistant to four antibiotic classes or carbapenemase-producing Enterobacteriaceae, according to the German guideline.Comparison of the two guidelines indicated that the definition of GN-MDRO, which is important for planning specific hygiene measures, differs between the Dutch and the German guidelines. Tables Enterobacteriaceae, Acinetobacter , and Pseudomonas aeruginosa. The Dutch guideline also defines multidrug-resistant variants of Stenotrophomonas maltophilia.On species level, these subgroups comprise P. aeruginosa.Antibiotics that are considered relevant for the definition of multidrug-resistance are similar in both guidelines: piperacillin, cefotaxime, ceftazidime, quinolons (i.e. ciprofloxacin according to German guideline), carbapenems (i.e. meropenem and/or imipenem according to German guideline) or confirmation of carbapenemase production. Additionally, the Dutch guideline also takes into consideration ESBL-phenotype and phenotypic resistance to cotrimoxazole and aminoglycosides. The German guideline additionally considers cefepime in case of If defined as GN-MDRO, hygienic measures, which exceed standard precautions, have to be applied in order to prevent spread in the hospital according to both guidelines. Depending on the type of microorganism, these special precaution measures may differ. This is shown in Table The German guideline advises single-room isolation of the patient on all wards of the hospital, whenever carriage or infection with a GN-MDRO classified as “4MRGN” is detected. For patients affected by GN-MDRO classified as “3MRGN” single-room isolation is required on high-risk wards only. Single-room isolation is always accompanied by wearing a disposable gown (when entering the room), gloves and, if indicated , a surgical mask. Patients carrying 3MRGN can be seen with standard hygiene measures at non-high-risk wards. High risk-wards are defined by local infection control personnel in every hospital, but should comprise e.g. intensive care units, including neonatology and haemato-oncology wards according to the guideline.Acinetobacter spp.. Strict isolation includes a single-room with ante-room, negative air pressure of the isolation room, wearing a disposable, long-sleeved gown, gloves and an FFP1 (filtering facepiece particle) mask. For the other GN-MDRO “contact isolation” is stipulated. Just like in Germany, this comprises wearing a disposable gown (before contact with the patient), gloves (when touching the patient and the patient’s immediate environment) and, if indicated , a FFP1 mask. However, many hospitals in the Netherlands perform “contact isolation” in single-rooms (without ante-room) for ESBL-Klebsiella and Carbapenem resistant Enterbacteriaceae (CRE), despite the fact that single-room isolation is only “suggested” but not mandatory, according to the guideline.In The Netherlands, isolation measures for “BRMO’s” are to a certain degree comparable, but show some differences. “Strict isolation” will be used for patients colonized or infected with a highly resistant According to the German guideline screening for 4MRGN carriage should be performed on all hospital wards for the following risk persons: 1.) contact with healthcare facilities in endemic countries; 2.) contact with a 4MRGN positive patient (same room). For these risk persons, pre-emptive isolation until the exclusion of 4MRGN is recommended. In the Netherlands, this policy of “search & isolate” is used for all GN-MDRO, has a few additional risk groups and is extended with a few Gram-positive MDRO, such as vancomycin-resistant enterococci and penicillin-resistant pneumococci. Consequently, these patients are screened and isolated until proven-negative, for all of the above mentioned MDROs.The conclusions of the Workshop on “ESBL/Multidrug Resistant Gram-negatives (“MRGN”) harmonization of the new guidelines in Germany and The Netherlands” in Nijmegen are illustrated in Table Enterobacteriaceae requires testing for certain mechanisms causing antibiotic resistance, i.e. ESBL and carbapenemase pheno- or genotypes have to be determined in line with routine microbiological diagnostics. The German guideline makes use of a newly created classification scheme (MRGN classification), based on phenotypic characterization and intended to guide infection control measures. This guideline is not meant to substitute reporting of resistance mechanisms detected by the laboratory, usually following the recommendations of EUCAST or CLSI standards. The MRGN classification grades multidrug-resistance by severity into 3MRGN and 4MRGN. For the correct classification as 3MRGN or 4MRGN, detection of specific resistance mechanisms like ESBL production is not required by the German guideline. The definition of GN-MDRO is mostly based on phenotypic susceptibility patterns, because the German guideline argues, that generally the transmission of resistant bacterial clones should be prevented whether the resistance mechanism is encoded on a plasmid or the chromosome. Detection of carbapenemase production, however, is essential, since according to the German guideline carbapenemase production in Enterobacteriaceae leads to classification as 4MRGN irrespective of other susceptibility test results.We identified that the Dutch and German guidelines show important discrepancies regarding the definition of GN-MDRO. In the Dutch guideline, defining GN-MDRO ampC is missed by the Dutch strategy.A major advantage of testing samples for presence of defined resistance determinants is a better understanding of resistance spread, especially if resistance is conferred by mobile genetic elements. A disadvantage might be that it is difficult to cover all mechanisms causing multidrug resistance by this approach; exemplarily, chromosomally- or plasmid-encoded Enterobacteriaeceae. Whereas special hygiene measures are required for all patients with ESBL-producing enterobacteria in The Netherlands, the German guideline recommends special precautions only for those cases in which patients are colonized or infected with strains showing co-resistance to ciprofloxacin (“3MRGN”). This difference is relevant: In Germany, the national surveillance system for antibiotic resistance (https://ars.rki.de) has published that among E. coli derived from inpatients, 10.8% were overall resistant to third-generation cephalosporines (indicative for ESBL) [E. coli isolates; i.e. 71% of third-generation cephalosporine-resistant E. coli isolates were ciprofloxacin resistant. In consequence, special hygiene precautions are estimated to be not applied for about 29% of patients with ESBL-producing E. coli in this example.Besides diagnostic issues, the main difference between the Dutch and German guideline is the divergent evaluation of ESBL-producing or ESBL)  in 2012.or ESBL)  of the EIn addition, co-resistance to aminoglycosides and quinolones, which defines GN-MDRO among enterobacteria according to the Dutch guideline, is not considered in this context in hospitals on the German side of the border. This might be due to the fact that aminoglycosides are markedly less used in German than in Dutch hospitals.In consequence, the fact that the definitions for GN-MDRO are different in the two current guidelines may lead to inefficient information transfer and different hygienic precaution measures when transferring a patient across the border. Therefore, the usage of a harmonized terminology would improve the possibilities for infection prevention, treatment and patient safety. Moreover, data on the prevalence of GN-MDRO in the border region should be reported in a way that they are directly comparable. Hence, the expert workshop revealed that harmonization of diagnostics report definitions is essential to facilitate cross-border cooperation between healthcare institutions. The initiation of cohort studies is warranted to yield comparable regional data.A. baumannii. This is an important similarity of both guidelines. As the prevalence of carbapenem-resistance is still <1% for enterobacteria in both The Netherlands and Germany, infection control guidance should clearly focus on preventing dissemination of these GN-MDRO as transmission was recently observed in Italian healthcare facilities [Enterobactericeae and Acinetobacter, for whom screening should be considered. However, the need to ensure prevention of carbapenem-resistant Enterobacteriaceae and Acinetobacter in the border region is also underpinned by recent investigations indicating the more wide-spread [With respect to recommendations on hygiene standards, both guidelines recommend single-room isolation of patients with carbapenem-resistant or carbapenemase-producing enterobacteria and cilities . In thise-spread  disseminproducing Enterobacteriaceae the Dutch guideline is more “microbiology-driven” compared with the German one. If ESBL is detected, such precautions are recommended. The German guideline advises single-room isolation on high-risk wards, because until now there is no general evidence for a benefit from single room isolation for 3MRGN E. coli in endemic areas. But on high risk wards (e.g. intensive care units), ESBL-producing and ciprofloxacin-resistant Enterobacteriaceae are likely to be transmitted to patient groups susceptible to acquire infections rather than colonization with these GN-MDRO.Regarding the use of special precautions for ESBL-Enterobacteriaceae including 3MRGN pathogens are already wide-spread in the general population. In The Netherlands ESBL-carriage was demonstrated in 8-10% of community patients [E. coli and Klebsiella, due to the differences in their molecular epidemiology and spread; multi-clonal/rarely spreading versus monoclonal and spreading, respectively [A clear problem related to these recommendations is that there is rising evidence that ESBL-producing patients , in Germpatients . Hence, ectively . Even thThe attendants of the workshop expressed that gathering regional data and data comparable between the two sides of the Dutch-German border is essential, because first, as a result of demographic changes in the border region, the proportion of population seeking medical treatment will rise including the proportion of those seeking treatment abroad. Second, particularly in the border region between two European high-income countries with cost-extensive healthcare systems, the number of highly susceptible individuals like neonates, immuno-compromised patients and elderly patients continues to increase due to implementation of advanced technologies. Together with an enhanced use of medical devices and implants vulnerable to bacterial contamination, hospital acquired infections are expected to rise, especially in the border region.Development of new diagnostic technology for rapid identification and personalized therapy and infection prevention management seems also very relevant to the attendants.Therefore, delivering innovative medical care and enabling “Healthy Ageing” will demand an increased level of infection control. This includes the necessity to cooperate across borders between countries (The Netherlands vs. Germany) as well as inter-sectorial borders  to a much larger extent than this is done nowadays. So far intersectorial cooperation has been focused on MRSA- management. The approach has to be stretched to all MDROs. The same holds for transborder cooperation in euregional networks including the public health structures (GGD/ÖGD) on both sides of the border. As previous studies performed within the Interreg IVa project EurSafety Health-net already demonstrated that synchronised strategies for screening of risk patients and standards of care in the framework of regional networks, were able to decrease MRSA prevalence , this sh


Visceral Leishmaniasis (VL) is an important protozoan opportunistic disease in HIV patients in endemic areas. East Africa is second to the Indian subcontinent in the global VL caseload and first in VL-HIV coinfection rate. Because of the alteration in the disease course, the diagnostic challenges, and the poor treatment responses, VL with HIV coinfection has become a very serious challenge in East Africa today. Field experience with the use of liposomal amphotericin B in combination with miltefosine, followed by secondary prophylaxis and antiretroviral drugs, looks promising. However, this needs to be confirmed through clinical trials. Better diagnostic and follow-up methods for relapse and prediction of relapse should also be looked for. Basic research to understand the immunological interaction of the two infections may ultimately help to improve the management of the coinfection. Visceral leishmaniasis (VL) is a vector-borne protozoan infection targeting the reticuloendothelial system Historically, East African VL has claimed the lives of many people, with the most infamous epidemic reported from South Sudan by Seaman et al. Leishmania almost always leads to a “deadly gridlock,” as they both have the same deleterious effect on the immune response The simultaneous infection of humans by HIV and The prevalence of HIV increased alarmingly from the mid-1980s to the 1990s in most African countries. Since 2000, however, a decline has been seen in the number of new HIV infections, which can be explained by a variety of factors, most notably preventive measures and access to ART Leishmania-nonimmune highlanders go to the VL-endemic regions, they become exposed and infected. Internal migration is also a risk factor for HIV, and those infected with HIV will develop overt disease (VL) more rapidly than those who are not infected The particularly high HIV coinfection rate in northwest Ethiopia could be due to the massive population movement in the region In Sudan, a hospital-based study in Khartoum reported a coinfection rate of 9.4% in 2002. Reports from southeast Sudan (Gedarif state), a region characterised by population movements, found HIV coinfection in 3.6% of VL cases in 2003 Leishmania donovani and is transmitted by a sand fly of the species Phlebotomus orientalis or P. martini in a predominantly anthroponotic cycle  VL in East Africa is caused by Leishmania parasites, which are normally concentrated in the reticuloendothelial organs, are often disseminated, and high parasite loads can be found in peripheral locations such as the skin, gut, lungs, peripheral blood, peritoneal fluid, and other organs and glands. Most of these atypical manifestations have been described in Europe with L. infantum as the etiologic agent Patients with VL typically present with prolonged fever, weight loss, prostration, splenomegaly, and pancytopenia Atypical presentations of VL can easily be confused with several other opportunistic conditions that occur in HIV patients and therefore cause a diagnostic challenge. Oral and skin lesions might be misdiagnosed as Kaposi sarcoma or skin tumours. Patients with disseminated disease often have a very weak immune response and may die even before the diagnosis is established. In resource-poor settings where case detection and management according to the existing guidelines are based on clinical case definitions and a syndromic approach L. donovani-transmission regions, a number of patients develop a maculopapular or nodular skin rash following successful treatment for VL. This syndrome, called post-kala-azar dermal leishmaniasis (PKDL), starts around the paranasal and perioral areas and spreads to the rest of the body. PKDL is reported in up to 50% of VL-treated patients in Sudan L. major, L. tropica, and L. aethiopica) requires confirmation by molecular tests.In The commonly available serological tests in the region, the rK39 rapid diagnostic test (RDT) and the direct agglutination test (DAT), showed inconsistent and generally lower performance in comparison to the Indian subcontinent in immunocompetent VL. A meta-analysis examining the diagnostic performance of the rK39 RDT reported an average sensitivity of 79% and specificity of 85% Studies on other serological tests , on urine antigen, and on molecular diagnosis are hardly available Few studies have assessed the performance of the main serological tests in HIV-positive patients in East Africa. Compared to HIV-negative individuals, sensitivity of the rK39 RDT (DiaMed-IT LEISH) was lower (77% versus 87%) among parasitologically confirmed cases in a study in Ethiopia. The sensitivity of DAT was generally higher but still lower among HIV-coinfected patients (89% versus 95%). Of interest, rK39 and DAT combined in a serial algorithm yielded a sensitivity of 98%, probably due to partly nonoverlapping sensitivity of both tests L. infantum have shown promising results when using concentrated blood (sensitivity 78% and specificity 100%) http://clinicaltrials.gov/show/NCT01360762).The suboptimal sensitivity of the commonly available serological test (rK39 RDT) explains the continued reliance on parasitological tests that can only be provided in a limited number of hospitals in the endemic areas. Some studies suggest that higher tissue parasite densities occur in HIV-coinfected patients Molecular testing  has been used in first-line diagnosis for VL in HIV-coinfected individuals in European countries and Brazil and merits further exploration in East Africa Diagnosis: Current knowledge and practiceL. donovani-endemic regions (Indian subcontinent)Lower accuracy of rK39 RDT and DAT in East Africa in the general VL patient population compared to other The limited data on HIV-coinfected individuals suggest somewhat lower accuracy of serological testsSequential diagnostic algorithms combining serological (rK39 followed by DAT) and parasitological testing achieve high accuracy with less need for invasive proceduresDiagnosis in HIV-coinfected patients still often relies on invasive procedures for parasitological diagnosis and for monitoring of treatment responseDiagnosis: Ongoing or planned VL diagnostic studies in HIV-coinfected individuals (Ethiopia)Noninvasive parasitological diagnosis using peripheral blood microscopyMicroculture inoculation of peripheral blood mononuclear cellsUrine antigen tests: Evaluation of KAtex for diagnosis and treatment response, test of cure (TOC)Molecular methods: reverse transcription loop mediated isothermal amplification (RT-LAMP) assay to be evaluated in 2013 (Foundation for Innovative New Diagnostics [FIND])L. donovani in East Africa than in the Indian subcontinent. Of interest, within East Africa, clear differences in efficacy were seen with these drugs in between and within different countries, with the lowest cure rates noted in northern Ethiopia and Sudan. Possibly, these observations could extend to VL-HIV coinfection as well.Striking differences exist in VL treatment response in the general VL patient population across and within regions For decades, antimonials have been the cornerstone of VL treatment in Africa. Although these drugs still maintain good efficacy in East Africa, their use is associated with unacceptably high and potentially fatal toxicity in VL-HIV coinfection In the search for a safer alternative, liposomal amphotericin B has increasingly been explored in East Africa. While studies have consistently reported an excellent tolerability, cure rates in HIV-infected individuals have been rather disappointing in this continent. At a total dose of 30 mg/kg, around 16% of primary VL and 56% of VL relapse cases demonstrate parasitological failure in northern Ethiopia Miltefosine has been evaluated in only one clinical trial in Ethiopia Whereas the combination of antimonials and paromomycin (for 17 days) is now recommended by WHO as first-line treatment in immunocompetent individuals in East Africa, experience with it as a first-line treatment for HIV-coinfected individuals is limited Given the high rates of lost to follow-up in most reported studies (often above 50%) L. infantum in Europe, suggested that secondary prophylaxis could reduce the risk of relapse in VL-HIV coinfection by at least 50% http://clinicaltrials.gov, NCT01360762).A recent systematic review, mainly containing data on Within Europe, the widespread use of ART in VL-endemic areas has had a major epidemiological effect on VL-HIV coinfection, with a pronounced reduction of new cases and prolonged disease-free survival for established VL-HIV coinfection. The effect on relapse has been more modest, with an estimated 50% reduction. Although ART has now been scaled up in most African countries, access to ART in the generally remote and underresourced VL-endemic areas remains challenging. Only in Ethiopia do all (major) VL treatment sites provide ART. Usually, ART is initiated as soon as the patient is clinically stable, typically during the second week after initiation of VL treatment.There is only one retrospective study on the effect of ART on survival in VL-HIV coinfection in East Africa, which did not demonstrate an effect Leishmania parasite on the immune system could relate to this requires further study. The lack of a clear case definition of IRIS in VL-HIV could further add to this lack of reporting. In East Africa, a few cases of VL have been reported after ART initiation (including seroconversion), but it is not clear to what extent these are true “unmasking” IRIS cases Overall and in clear contrast with TB-HIV, the immune reconstitution inflammatory syndrome (IRIS) appears to be an exception in VL-HIV coinfection. Only a handful of clear-cut case reports of (ART-associated) VL-IRIS have been reported at the global level Leishmania. One potential approach could consist of using PI-based ART instead of non-nucleoside reverse transcription inhibitor (NNRTI)-based ART in VL-HIV-endemic regions. However, with detailed animal or clinical studies lacking, additional research is required before HIV-1 PIs should be taken forward towards this goal With the global scaling-up of ART, HIV-1 protease inhibitors (PIs) are increasingly available in VL-HIV-endemic regions, including regions in Africa. Several lines of evidence suggest that HIV-1 protease inhibitors might directly exert antiparasitic effects, including effects against The infectiousness of HIV-coinfected patients to sand flies in endemic areas of anthroponotic foci has not been studied. However, coinfected patients were found to have higher tissue parasite loads and higher rates of PKDL Leishmania infection in HIV patients was recognized late. The poorest segments of the population, such as migrant daily laborers, are affected. Thus, the diagnostic and treatment challenges of VL-HIV coinfection have continued to date, especially in East Africa As a neglected disease, the significance of The control of VL requires a combined effort at vector control, improved living standards and case detection, and early treatment Case detection and management is especially challenging for HIV-infected persons. To have a larger impact on VL-HIV coinfection, a comprehensive and multipronged approach will be required. Better diagnostic and curative options would help to improve case detection and patient management. As with other HIV-associated coinfections , preventive strategies should be explored. As the experience in Europe testifies, the preventive effect of large-scale introduction of ART merits exploration. Although the current WHO guidelines now recommend early ART initiation Diagnosing VL in HIV patients relies mainly on parasite detection from tissue aspiration. This is due to the insufficiently sensitive serological tests that also do not help to diagnose relapsed cases, which are very common in HIV patients. However, tissue aspiration is associated with potential risk of fatal bleeding that requires experience and health facilities equipped to handle this potential complication. Thus, a simple-to-use but reliable antigen-detection diagnostic procedure or tool that can be applicable for treatment monitoring and diagnosing relapses is urgently required.For VL-HIV case management, field experience favors the use of the following combination treatment: liposomal amphotericin B and miltefosine Tackling VL-HIV coinfection in the long run, along with combating VL in general, will strongly depend on the strength and commitment of the national programs. However, to date, the national programs still rely on substantial external support in most East African countries. Moreover, a strong link with the national HIV program will be needed for efficient and integrated program management. Though the HIV prevalence is declining in Ethiopia, the unstable socioeconomic and political situations in South Sudan and the high population migrations in the region warrant continuous effort for the control of both diseases.Northwest Ethiopia has the highest known burden of VL-HIV coinfection rates in the world.VL-HIV coinfection is associated with diagnostic and treatment challenges that still need additional research.Atypical clinical presentations and poor performance of rapid serological tests among the HIV co-infected VL patients poses a diagnostic challenge.There is a high rate of treatment failure and relapse of VL among HIV-coinfected patients.Combination treatment with liposomal amphotericin B and miltefosine followed by secondary prophylaxis and ART seems to be a promising standard of care that needs clinical trials.Hurissa Z, Gebre-Silassie S, Hailu W, Tefera T, Lalloo DG, et al. (2010) Clinical characteristics and treatment outcome of patients with visceral leishmaniasis and HIV co-infection in northwest Ethiopia. Trop Med Int Health 15: 848–855. doi: 10.1111/j.1365-3156.2010.02550.xter Horst R, Tefera T, Assefa G, Ebrahim AZ, Davidson RN, et al. (2009) Field evaluation of rK39 test and direct agglutination test for diagnosis of visceral leishmaniasis in a population with high prevalence of human immunodeficiency virus in Ethiopia. Am J Trop Med Hyg 80: 929–934.ter Horst R, Collin SM, Ritmeijer K, Bogale A, Davidson RN (2008) Concordant HIV infection and visceral leishmaniasis in Ethiopia: the influence of antiretroviral treatment and other factors on outcome. Clin Infect Dis 46: 1702–1709. doi: 10.1086/587899Ritmeijer K, ter Horst R, Chane S, Aderie EM, Piening T, et al. (2011) Limited effectiveness of high-dose liposomal amphotericin B (AmBisome) for treatment of visceral leishmaniasis in an Ethiopian population with high HIV prevalence. Clin Infect Dis 53: e152–e158. doi: 10.1093/cid/cir674Ritmeijer K, Dejenie A, Assefa Y, Hundie TB, Mesure J, et al. (2006) A comparison of miltefosine and sodium stibogluconate for treatment of visceral leishmaniasis in an Ethiopian population with high prevalence of HIV infection. Clin Infect Dis 43: 357–364. doi: 10.1086/505217


Chiroptera: Pteropodidae) are the natural host of Hendra virus (HeV) which sporadically causes fatal disease in horses and humans in eastern Australia. While there is strong evidence that urine is an important infectious medium that likely drives bat to bat transmission and bat to horse transmission, there is uncertainty about the relative importance of alternative routes of excretion such as nasal and oral secretions, and faeces. Identifying the potential routes of HeV excretion in flying-foxes is important to effectively mitigate equine exposure risk at the bat-horse interface, and in determining transmission rates in host-pathogen models. The aim of this study was to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. A total of 2840 flying-foxes from three of the four Australian mainland species  were captured and sampled at multiple roost locations in the eastern states of Queensland and New South Wales between 2012 and 2014. A range of biological samples  were collected from anaesthetized bats, and tested for HeV RNA using a qRT-PCR assay targeting the M gene. Forty-two P. alecto (n = 1410) had HeV RNA detected in at least one sample, and yielded a total of 78 positive samples, at an overall detection rate of 1.76% across all samples tested in this species (78/4436). The rate of detection, and the amount of viral RNA, was highest in urine samples , identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. There were no detections in P. poliocephalus  or P. scapulatus , suggesting (consistent with other recent studies) that these species are epidemiologically less important than P. alecto in HeV infection dynamics. The study is unprecedented in terms of the individual animal approach, the large sample size, and the use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.Pteropid bats or flying-foxes ( Henipavirus first described in September 1994 in Australia, when it was identified as the aetiological agent in an outbreak of acute equine respiratory disease that resulted in the deaths of 14 horses and 1 human, in the Brisbane suburb of Hendra [Chiroptera: Pteropodidae), which were identified as the likely primary wildlife reservoir through a series of serological and viral isolation studies [P. alecto), Grey-headed flying-fox , Spectacled flying-fox (P. conspicillatus) and Little red flying-fox (P. scapulatus) [Hendra virus (HeV) is a paramyxovirus of the genus f Hendra . The foc studies –5. Antibpulatus) .P. poliocephalus yielded inconsistent results; most infected animals seroconverted, but positive immunohistochemistry (IHC) findings were rarely observed, and virus isolation was mostly unsuccessful in a diverse range of tissues [P. poliocephalus where live virus was recovered, vertical transmission was also demonstrated by the isolation of virus from the foetal spleen and kidney [P. poliocephalus, and from the foetal lung of a pregnant P. alecto [P. alecto essentially replicated earlier findings, with only 10 of the 20 HeV-inoculated animals seroconverting, negative IHC results in all tissues, and little evidence of HeV infection on histopathological examination of tissues post-mortem [P. alecto on days 6 and 8 post-inoculation, albeit at low titres at the limit of detection, and with no evidence of HeV antigen on IHC staining in either the bladder or kidney post-mortem [Initial experimental HeV transmission studies with  tissues , 8. In o. alecto . A more t-mortem . Halpin t-mortem . All expWhile the apparent lack of clinical disease attributable to HeV infection in flying-foxes is consistent with coevolution theories pertaining to intimate host-pathogen relationships , 12, theThe problem with extrapolating the results of experimental HeV infection studies to likely modes of transmission between wild flying-foxes, or indeed from wild flying-foxes to equine spillover hosts, is the extent to which experimental infection models truly reflect the biology of HeV in naturally-infected flying-foxes. While experimental infection studies can yield useful information, they are often subject to important caveats , and their direct relevance to gaining an accurate understanding of natural disease systems may be questionable . AdvanceP. alecto [The importance of understanding the main routes of HeV excretion in naturally-infected flying-foxes can be viewed on a number of levels. While we have strong evidence that urine is an important infectious medium that likely drives bat to bat transmission, and in all likelihood bat to horse transmission, we are less certain about the relative importance of alternative routes of excretion , notwithstanding a small number of detections in oral and rectal swabs in both experimentally and naturally-infected . alecto , 22. If,. alecto , and whi. alecto , they woThe primary aim of this study was to determine the major routes of HeV excretion in flying-foxes. A secondary aim was to identify any between-species variation in excretion prevalence. The expectation is that the findings will more precisely inform equine risk mitigation strategies and add to our understanding of the relative risk of HeV infection at the flying-fox species level.Fieldwork in all locations was approved under the Queensland Department of Agriculture, Fisheries and Forestry Animal Ethics Committee Permit SA 2011/12/375 and the Queensland Department of Environment, Heritage and Protection Scientific Purposes Permits WISP05810609 and WISP14100614. Full details of capture and sampling methods are presented below. All methods represent current best practice and were specifically approved under the permits. Capture and sampling was undertaken by trained and experienced teams including a veterinarian. No animals were sacrificed.Flying-foxes were sampled at multiple roost locations in the eastern Australian states of Queensland (QLD) and New South Wales (NSW) between February 2012 and June 2014. The study utilised capture events associated with two related studies: a cross-sectional study series investigating the association between flying-fox roost disturbance and HeV infection dynamics ; and a sP. alecto were preferentially targeted to address the objectives of the related longitudinal study. From December 2013, where the total daily capture limit was not met by P. alecto in any capture event, the balance was made up with P. poliocephalus and P. scapulatus selected to better balance overall sex and age classes.Flying-foxes were captured pre-dawn, as they returned to the roost, in 18 m mist nets suspended between either 12 m aluminium poles or 20 m fibreglass telescopic poles (Spiderbeam GmbH). Animals were carefully extracted from the mist net and immediately placed in a pillow-case tied to a horizontal line, which enabled bats to roost in a natural inverted position while awaiting sampling. All flying-foxes were anesthetised for the collection of samples, as described by Jonsson et al. (2004) . SpeciesFifty μL of urine, serum, urogenital, preputial, nasal, oral and rectal swab samples were directly added to 130 μL of lysis solution  to inactivate virus particles and preserve RNA for PCR screening. Total nucleic acid was immediately extracted from urine, serum, urogenital and preputial swab samples in lysis buffer using a magnetic bead based nucleic acid extraction kit  run on a magnetic particle handling system  according to the manufacturer’s instructions. The preserved nasal, oral and rectal swab samples were held at 4°C for 24–48 hours, before being stored at -80°C prior to testing several weeks later.P. alecto, P. poliocephalus and P. scapulatus captured at multiple sampling locations and across multiple times.Urine, serum, urogenital and preputial swabs were used as an initial screening panel for all individual flying-foxes. Subsequently, the full complement of samples was tested from all HeV-positive flying-foxes, including the packed haemocyte sample retained after separation of serum. We also tested a subset of all available samples from all three species to ensure that possible additional routes of HeV excretion  weren’t being overlooked in the initial screening approach. The practical outcome of this process was to test the full complement of samples from an essentially random selection of The AgPath-ID One-Step RT-PCR Kit (Life Technologies) was used for qRT-PCR, with 5uL of nucleic acid extract added to 20 μl of mastermix. Forward and reverse primers and probe that target a 69 base pair region on the M gene were used, as described by Smith et al. (2001) . PositivA total of 2840 individual flying-foxes were captured and sampled across 10 roost sites over the 28-month period . Of thesP. alecto, and all 42 HeV-positive P. alecto were captured at Boonah between June 2013–2014. The majority of HeV-positive flying-foxes had HeV RNA detected in a single sample , while a smaller number of animals had HeV RNA detected in multiple samples . One individual flying-fox had HeV RNA detected on the full panel of samples available for testing .Of the three flying-fox species sampled during this study, HeV RNA was detected only in P. alecto from which a urine sample was successfully collected, 26 (i.e. 90%) had HeV RNA detected in their urine, at Ct values ranging from 22.26–37.33 , with less frequent detections urogenital and preputial swabs  and serum  . There were no detections in P. poliocephalus  or P. scapulatus   . The 42 samples) .P. alecto is higher than the 0.8% HeV detection rate reported by Breed et al. (2013), who sampled 124 P. alecto in Australia (n = 64), Papua New Guinea (n = 15) and Indonesia (n = 45) and detected one HeV-positive animal, using the same qRT-PCR for detection of HeV RNA, and with a comparable selection of screening samples . The single HeV-positive P. alecto described by Breed et al. (2013) was a sub-adult male, with detection of HeV RNA in blood, urine and saliva [This study represents the single largest sampling of individual wild-caught flying-foxes for HeV surveillance in Australia, and more broadly, for henipavirus surveillance globally. While other studies have used serological tools as a proxy for infection status of individual flying-foxes –31 to esd saliva . The Ct One point of difference between our study and Breed et al. (2013) which may contribute to the difference in crude prevalence estimates is the physical choice of swab; the patented flocked swabs utilised in our study are purportedly more sensitive for bacterial and viral diagnostics than the cotton-tipped swabs used by Breed at al. (2013) –34. Our Our study confirms that urine is the most important route of HeV excretion in naturally infected flying-foxes –21, and P. alecto). Given the successful isolation of HeV from pooled urine samples collected beneath Australian flying-fox roosts [P. alecto [P. alecto (28–29 units in select throat swabs and urine samples) [P. alecto in this study indicate that free-living flying-foxes are indeed capable of excreting potentially infectious viral loads. While previous virus isolation attempts from pooled flying-fox urine samples collected under-roost showed no apparent correlation with lower Ct values [P. alecto shedding large amounts of potentially infectious virus in this excretory medium. In contrast to pooled urine samples, interpretation of the Ct value of individual urine samples is not complicated by pooling, samples are collected directly, and samples are immediately placed on ice. Future studies that pursue virus isolation from such samples would readily establish any correlation, although the BSL-4 pathogen status of HeV imposes significant logistical constraints to undertaking virus isolation experiments.HeV-positive urine samples had comparable mean and minimum Ct values to HeV-positive urogenital swabs and rectal swabs. Mean and minimum Ct values in oral and nasal swabs were higher, indicating relatively lower levels of viral RNA associated with these portals. Although virus isolation was not attempted on any of the HeV-positive samples collected in this study, the use of Ct values as a semi-quantitative proxy for likely ‘viral load’  is considered reasonable  closely followed by systemic (i.e. haematogenous) spread to internal organs  [The detection of HeV RNA in serum of naturally-infected kidneys) , 9, 37. kidneys) .P. alecto clustered in April–June, which coincides with early to mid-gestation in this species . Pregnancy has previously been recognised as a risk-factor for HeV infection in P. scapulatus and P. conspicillatus, using HeV serology as a proxy for infection status [P. alecto , although only one of these females was pregnant during the experimental period [Latent HeV infection interspersed with periods of recrudescence has also been hypothesised as a potentially important mechanism in maintaining persistent infection at both the individual bat and flying-fox population level , 39, andn status , 30, andl period , 40, temP. poliocephalus and P. scapulatus adds to a growing body of evidence suggesting that P. alecto and P. conspicillatus are the main reservoir host species for HeV [P. scapulatus in this study may simply reflect the relatively limited, non-targeted sampling effort in this species , or alternatively, the narrow spatiotemporal sampling of P. scapulatus given its extensive geographic occurrence and highly nomadic nature [P. scapulatus [P. scapulatus is not a primary reservoir host.The complete lack of detections in  for HeV , 41. It c nature . Yet, wiapulatus , we werepoliocephalus is unambiguous given the predominant structured temporal surveillance at two disparate locations, and the large sample size (n = 1168). At the Boonah roost, which yielded the majority of the P. poliocephalus samples, all 42 HeV detections were made in co-roosting P. alecto in the same time period. At the Sydney Royal Botanic Gardens roost, the March–May 2012 sampling period encompassed breeding and early gestation in P. poliocephalus [P. poliocephalus has a significantly lower infection rate than P. alecto. Our findings in relation to both P. scapulatus and P. poliocephalus are consistent with several other recent studies employing different sampling methodologies [Interpretation of the negative HeV data for P. cephalus . The laccephalus , 30, 44 ize n = 18. At thedologies , and proP. poliocephalus has previously led authors to conclude that these potential excretory routes may be of little importance [P. alecto. Collectively these results suggest that flying-fox transmission likely revolves around direct contact with infectious urine of conspecifics (or possibly cross-species transmission in multi-species roosts). Hypothesised indirect transmission pathways facilitating spillover to horses via urinary contamination of pasture, feed and water sources remain plausible [In terms of possible viral transmission pathways between flying-foxes, and from flying-foxes to horses, the results of this study provide new empirical evidence on the most likely sources of HeV exposure for both flying-foxes and horses. While failure to isolate HeV from nasal, oral, rectal and urinary samples from experimentally-infected portance , a more portance , 44. Theportance , 8 has aportance , 30, andportance . The rouportance , 21, 25 lausible , 46. Thilausible , 44, andlausible  elaboratlausible . AdditioP. poliocephalus and P. scapulatus strongly suggest these species are epidemiologically less important than P. alecto. The study is unprecedented in terms of our individual animal approach, our large sample size, and our use of a molecular assay to directly determine infection status. These features provide a high level of confidence in the veracity of our findings, and a sound basis from which to more precisely target equine risk mitigation strategies.We sought to identify the major routes of HeV excretion in naturally infected flying-foxes, and secondarily, to identify between-species variation in excretion prevalence. We captured free-living flying-foxes over space and time, and collected and screened multiple samples from each individual using a molecular approach to identify HeV RNA. The rate of detection, and the amount of viral RNA was highest in urine samples , identifying urine as the most plausible source of infection for flying-foxes and for horses. Detection in a urine sample was more efficient than detection in urogenital swabs, identifying the former as the preferred diagnostic sample. The detection of HeV RNA in serum is consistent with haematogenous spread, and with hypothesised latency and recrudesence in flying-foxes. The lack of detections in


Klebsiella spp. are of major concern. To control transmission, deep understanding of the transmission mechanisms is needed. This systematic review aimed to identify risk factors and sources, clonal relatedness using molecular techniques, and the most effective control strategies for ESBL-producing Klebsiella spp. A systematic search of PubMed, Embase, and Outbreak Database was performed. We identified 2771 articles from November 25th, 1960 until April 7th, 2014 of which 148 were included in the systematic review and 23 in a random-effects meta-analysis study. The random-effects meta-analyses showed that underlying disease or condition  generated the highest pooled estimate. ESBL-producing Klebsiella spp. were spread through person-to-person contact and via sources in the environment; we identified both monoclonal and polyclonal presence. Multi-faceted interventions are needed to prevent transmission of ESBL-producing Klebsiella spp.Healthcare-related infections caused by extended-spectrum beta-lactamase (ESBL)-producing  Healthcare-related infections (HRIs) are a major clinical problem worldwide. In 2011, the World Health Organization (WHO) reported that in a mixed patient population the pooled HRI-prevalence was 10.1% in low- and middle-income countries and 7.6% in high-income countries .Klebsiella spp. have been recognized as the most frequent cause of MDR-GNB outbreaks, particularly after the emergence of the extended-spectrum beta-lactamase (ESBL) enzymes ..12].th, 2014) to identify studies which examined the transmission of multidrug-resistant (MDR) Klebsiella spp., identified potential risk factors, described modes of transmission, described laboratory methods used for the identification, and described the effective interventions to prevent transmission of MDR Klebsiella spp. with using the terms as applied in We searched PubMed, Embase, and the Outbreak Database -based techniques for typing. We assessed the result of molecular typing methods and calculated the total number of identified patterns. We defined a cluster as ≥ two similar patterns of ESBL-producing Klebsiella spp. isolates. Likewise, a unique isolate was defined as a single pattern. The term monoclonal presence referred to a single cluster and the term polyclonal presence referred to ≥ two clusters. We calculated the total number of patterns, the clusters including cluster sizes, and the single patterns. If the information was available, we performed the cluster analyses based on the number of patients, otherwise on the number of isolates. We also reviewed studies about infection control strategies and prevention programs. We assessed the standard interventions possibly combined with additional control strategies, and reported which were most successful strategies to stop transmission. We compiled data from two studies that were presented in four publications in the result section [In order to assess clonal relatedness and transmission patterns of ESBL-producing  section –18.P values <0.05 as statistically significant. Bias assessment plots were constructed to explore publication bias using the Egger and Begg-Mazumbdar  indicators [We combined all associated factors that reported an odds ratio (OR) and a 95% confidence interval (95% CI) into ten different categories: 1, medical devices ; 2, prior cephalosporin exposure; 3, prior quinolone exposure; 4, prior other antibiotic exposure; 5, prior antifungal exposure; 6, length of hospital stay; 7, patient characteristics (e.g. age); 8, underlying disease or condition ; 9, medical procedures ; 10, other (e.g. exposure to the hands of HCWs). Studies reporting associated factors for mortality were excluded. Random-effects meta-analyses were performed for all categories except the “prior antifungal exposure”, “medical procedures” which had less than three factors and “other” that comprised many various factors. Lytsy et al., used three different models of multivariable analyses to find reliable estimates for the most important variables. However, we chose to only include model 1 in our meta-analyses . We appldicators , 20.The methodological quality of all studies in the random-effects meta-analyses was assessed using the strengthening the reporting of observational studies in epidemiology (STROBE) guidelines (Table A and Table B in n = 59) particularly in France (n = 16), followed by Asia , North America , Africa , South America , multiple regions: Europe and Asia  and Australia  with 49 countries in total. All studies were published between 1991 and 2014 with study periods of less than a month up to seven years. Thirty-one studies (20.9%) indicated the suspected or identified index case. The study population was predominated by adult patients , followed by neonates , pediatric patients , and mix groups including the studies located in hospitals but which did not mention the study population . Seventy-six percent of all studies took place at the ICU (n = 113). These studies can be further divided in neonatal ICU , pediatric ICU , adult ICU  and mix ICU units . However, fifty-two studies were located both at ICU and non-ICU.We identified a total of 5,608 articles as potentially relevant when using our search strategy . Of thesKlebsiella spp. In addition, four studies identified the associated factors for bloodstream infections caused by ESBL-producing K. pneumonia  whereas the patients were the main reservoirs , followed by the hands of HCWs . Interestingly, one study showed food as a transmission source for ESBL-producing K. pneumonia [Fifty-seven studies identified environmental sources and/or surrounding reservoirs for ESBL-producing lla spp. . Contamineumonia .Klebsiella spp. and the analyses of genetic similarity  in seven studies. Multilocus sequence typing (MLST) was performed in seven studies as well as random amplified polymorphic DNA (RAPD). Nineteen studies performed more than one molecular method to analyze clusters. The average number of patients in the 120 studies was 22.5 (ranging from 1 to 295 patients). The median range of the number of patterns was 1 to 10 (median range of cluster-size medians from 1 to 15). In 111 studies that provided both number of clusters and the cluster size based on the number of patients, the median number of clusters was 2 ranging from 0 to 15 and the median of the cluster-size medians was 6 with a range of 0 to 81. In particular for K. pneumoniae, the median number of clusters was 2 and the median of cluster-size medians was 6. Further, the median number of clusters and the median of the cluster-size medians were 0 for K. oxytoca. Three surveillance studies reported an identical pattern indicating the clonality of identified ESBL-producing Klebsiella spp. strains in hospitals. In particular for the outbreak events, 43 studies showed monoclonal and 34 studies showed polyclonal presence of ESBL-producing Klebsiella spp. among patients within the hospital.One hundred-twenty studies described the molecular methods used to type ESBL-producing milarity . In partKlebsiella spp. transmission in hospitals should be followed by infection control strategies. However, not all studies provided detailed information regarding the interventions. We identified 84 studies that described the interventions during the study period. Twenty-eight studies reported the standard interventions that did not succeed. All but three studies described the additional and/or successful strategies to prevent the spread of ESBL-producing Klebsiella spp. in hospitals. Ultimately, we presented the top ten strategies to control ESBL-producing Klebsiella spp. in hospitals from 71 studies  and prior cephalosporin exposure  as the most significant associated factors for the presence of ESBL-producing Klebsiella spp. Consistent with our study, Cornejo-Juárez et al., indicated the increased risk of ESBL-Escherichia coli bacteremia in patients with hematologic malignancies who had prior cephalosporins exposure [Enterobacteriaceae in Latin America that reported prior antibiotic exposure, in particular cephalosporins as the associated risk factor with a statistically significant OR above one for the acquisition of ESBL-producing Klebsiella spp. [Klebsiella spp. can cause fatal outcomes in association with other associated risk factors [Klebsiella spp. In general, likewise in case of Methicillin-resistant Staphylococcus aureus, HCWs who are colonized with ESBL-producing Klebsiella spp. play a pivotal role in the transmission through their hands [Klebsiella spp. [This is the first systematic review and meta-analyses to identify the transmission pattern of ESBL-producing exposure . Our finlla spp. . Neverthlla spp. . Moreove factors . Our stuir hands . Even molla spp. , 35.Klebsiella spp. among patients [PFGE still is the gold standard for molecular typing, in particular to prove the transmission of ESBL-producing patients . Howeverpatients , 37. Furpatients . Howeverpatients . Studiespatients .Klebsiella spp., reinforcement of hand hygiene and an antibiotic control program were the most successful interventions. Firstly, hand hygiene is a simple and low cost intervention to prevent the presence of ESBL-producing Klebsiella spp. The introduction of hand antiseptic will reduce the contamination of the hands with of ESBL-producing Klebsiella spp. [Klebsiella spp. transmission.To stop the spread of ESBL-producing lla spp. . Recent lla spp. . Secondllla spp. . This adKlebsiella spp. Nonetheless, reporting the cases of ESBL-producing Klebsiella spp. according to STROME-ID guidelines can give clear information about the evidence to detect the transmission dynamics with molecular typing and helps in making the health-policy decision. In general, the studies that were included answered our research questions; however, our study indicated the importance to report structured articles that follow appropriate guidelines for a high study quality.Most studies included in the meta-analyses and cluster analysis had a low study quality using the recommended guidelines. However, in particular for the studies that were assessed with STROME-ID guidelines, they had wide range of key objectives that did not focus solely on molecular epidemiology of ESBL-producing Klebsiella spp. In the meta-analyses, we identified several risk factors that were associated with the presence of ESBL-producing Klebsiella spp. We also summarized the most successful interventions to prevent the spread of ESBL-producing Klebsiella spp., which to our knowledge has never been done before. However, our study also has some limitations. Firstly, the heterogeneity of all included studies in combination with the diverse models used in all individual statistical analyses, here combined in the meta-analyses. Therefore, we performed a random-effects model for the meta-analyses. Secondly, due to the different definitions of colonization and infection in the studies, we termed them as presence of ESBL-producing Klebsiella spp. As a consequence, reporting them separately was not possible. Thirdly, our study focused on clonal spread; hence, we did not include studies about plasmid transfers. Fourthly, some studies only performed molecular typing investigation on selected samples due to cost effectiveness. Therefore, most probably we have bias on the number of clusters. Fifthly, publication bias might have occurred since we have a broad range of articles from different objectives to comprehensively answer our research questions.The major strength of our study was the inclusion of a large number of hospitalized patients with ESBL-producing Klebsiella spp., which results in increased morbidity and mortality, may occur by either direct spread from patient to patient or indirect transmission via surrounding reservoirs and sources in the environment. Obviously, molecular typing techniques can identify transmission of ESBL-producing Klebsiella spp. within the hospital. Prior antibiotic exposure holds a key role for the presence of ESBL-producing Klebsiella spp. particularly cephalosporin use. Multi-faceted interventions, including reinforcement of hand hygiene and control of antibiotic use, are necessary to prevent the spread of ESBL-producing Klebsiella spp. Further studies on plasmid transfer are needed to learn more about transmission of ESBL-producing Klebsiella spp. within a hospital. In addition, it is important to report studies in a more structured way that systematically follows suitable guidelines.The presence of ESBL-producing S1 File(DOCX)Click here for additional data file.S2 File(DOCX)Click here for additional data file.S3 FileQuality assessment scores of the 23 articles that were included in the random-effects meta-analysis study (Table A). Quality assessment scores of the 23 articles that were included in the random-effects meta-analysis study, using the STROBE guidelines (Table B). Quality assessment scores of the 15 case-control studies that were included in the random-effects meta-analysis study, using the Newcastle Ottawa Scale (Table C1). Quality assessment scores of the three cohort studies that were included in the random-effects meta-analysis study, using the Newcastle Ottawa Scale (Table C2). Quality assessment scores of 120 articles that were included in the cluster analysis study (Table D).(DOCX)Click here for additional data file.