Comparison of Leishmania OligoC-TesT PCR with Conventional and Real-Time PCR for Diagnosis of Canine Leishmania Infection

There is a need for standardization and simplification of the existing methods for molecular detection of Leishmania infantum in the canine reservoir host. The commercially available OligoC-TesT kit incorporates standardized PCR reagents with rapid oligochromatographic dipstick detection of PCR products and is highly sensitive for use in humans but not yet independently validated for use in dogs. Here we compare the sensitivity of OligoC-TesT with those of nested kinetoplast DNA (kDNA) PCR, nested internal transcribed spacer 1 (ITS-1) PCR, and a PCR-hybridization protocol, using longitudinal naturally infected canine bone marrow samples whose parasite burdens were measured by real-time quantitative PCR (qPCR). The sensitivity of OligoC-TesT for infected dogs was 70% (95% confidence interval [CI], 63 to 78%), similar to that of kDNA PCR (72%; 95% CI, 65 to 80%; P = 0.69) but significantly greater than those of PCR-hybridization (61%; 95% CI, 53 to 69%; P = 0.007) and ITS-1 nested PCR (54%; 95% CI, 45 to 62%; P < 0.001); real-time qPCR had the highest sensitivity (91%; 95% CI, 85 to 95%; P < 0.001). OligoC-TesT sensitivity was greater for polysymptomatic and oligosymptomatic dogs than for asymptomatic dogs (93%, 74%, and 61%, respectively; P = 0.005), a trend also observed for the other qualitative PCR methods tested (P ≤ 0.05). Test positivity increased with increasing parasite burdens, as measured by real-time qPCR: OligoC-TesT and kDNA PCR detected 100% and 99% of positive samples when parasite burdens exceeded 74 and 49 parasites/ml, respectively. OligoC-TesT has high sensitivity for detection of canine Leishmania infections; its ease of operation and ease of interpretation are further advantages for veterinary diagnostic laboratories and for large-scale survey work in developing countries.Zoonotic visceral leishmaniasis (ZVL), a vector-borne disease caused by the protozoan parasite Leishmania infantum [Leishmania (Leishmania) infantum chagasi (11)], results in significant mortality and morbidity in the reservoir host (the domestic dog) and represents a serious public health problem in many regions where it is endemic (the Mediterranean basin, Latin America, and parts of central and eastern Asia). Serological methods are the most technically straightforward of the available tests for diagnosis of canine infection, but these methods lack sensitivity for asymptomatic and early-stage infections (5, 17, 24). Detection of Leishmania parasites in canine clinical samples has traditionally been performed by means of microscopic examination of stained tissue specimens and by parasitological culture, which are known to be insensitive. PCR for amplification of defined parasite DNA sequences is highly sensitive for animals with clinical disease and has higher sensitivity than serology for asymptomatic animals and early-stage infections (8, 9, 19, 23, 29). However, the technical complexity of PCR may reduce its practicality for use in developing countries most affected by ZVL. Furthermore, there is a lack of standardization in the selection of target Leishmania DNA sequences and experimental PCR protocols used in laboratories worldwide, which complicates objective comparisons of test sensitivity and specificity. In order to address some of these issues, a commercially available PCR test kit (Leishmania OligoC-TesT) has been developed and validated for detection of Leishmania parasite DNA in human specimens (3, 6). Sensitivities of the test ranged from 77.8% to 100% for clinical samples from patients with visceral leishmaniasis in Sudan and Kenya, with a limit of detection of 1 parasite in 180 μl blood (3). OligoC-TesT has not yet been validated independently for use in dogs. The aims of this study were therefore (i) to measure the sensitivity of OligoC-TesT compared with those of three conventional PCR procedures (nested PCR for amplification of kinetoplast DNA [kDNA], nested PCR of internal transcribed spacer region 1 [ITS-1] of the rRNA gene, and kDNA/rRNA PCR followed by hybridization with specific oligonucleotide probes) and with that of real-time quantitative PCR (qPCR), (ii) to compare the sensitivities of OligoC-TesT and the PCR methods listed above for samples from infected dogs that were positive or negative for clinical signs of leishmaniasis, and (iii) to determine the analytical sensitivity of OligoC-TesT relative to canine bone marrow parasite burdens measured by real-time qPCR. For this study, we used samples collected in a longitudinal study of a cohort of naturally L. infantum-infected domestic dogs in Brazil.     

Cellular and Humoral Responses to Leishmania major Virulence Factors in Healed Cutaneous Leishmaniasis and Mediterranean Visceral Leishmaniasis Patients

Cellular and humoral immune responses of healed cutaneous leishmaniasis and Mediterranean visceral leishmaniasis patients were evaluated against results for Leishmania major virulence proteins L. major protein disulfide isomerase (LmPDI) and mitogen-activated protein kinase kinase (MAPKK). Only MAPKK induces significant peripheral blood mononuclear cell proliferation with gamma interferon production as well as antibody responses. Thus, MAPKK may be of interest in Leishmania vaccination and serodiagnosis.The leishmaniases are diseases caused by vector-borne pathogens that represent a major public health problem affecting the lives of millions of people worldwide (http://www.who.int/whr/en). Depending on the parasite species and on the immunological response of the human host, leishmaniasis ranges from an asymptomatic infection to a self-limiting cutaneous lesion(s) or a fatal visceral form.No anti-Leishmania vaccine is available at the moment. Different studies showed that development of Th1- and Leishmania-specific cytotoxic immune responses correlate with healing of patients with cutaneous leishmaniasis (CL) (3, 12). An intense effort is being made to identify antigens that could induce an immune state similar to that developed by individuals who recover from symptomatic infection and are resistant to a subsequent natural challenge. Such antigens may contribute to the development of an anti-Leishmania vaccine.Diagnostic tools targeting leishmaniasis are available. However, parasite detection is invasive and poorly sensitive. Serological diagnosis using various techniques based on detection of Leishmania-specific antibodies that are developed during acute disease are often more sensitive, less time-consuming, and more user friendly (4, 11, 15).In an attempt to identify new antigens to be used as vaccines or for serodiagnosis, we focused on Leishmania virulence factors. Indeed, several studies described these factors as potentially immunogenic in humans, mice, and, more recently, dogs (6, 8, 9, 13). Here, cellular and humoral immune responses of healed CL (hCL) and Mediterranean visceral leishmaniasis (MVL) patients were evaluated against results for Leishmania major protein disulfide isomerase (LmPDI) and mitogen-activated protein kinase kinase (MAPKK), which we and others previously described as potential virulence factors (2, 10).We produced LmPDI (55 kDa) and MAPKK (40 kDa) in the prokaryotic expression pET system (Novagen, Gibbstown, NJ) and then purified them by affinity chromatography (Fig. ​(Fig.11).Open in a separate windowFIG. 1.Expression of recombinant proteins in Escherichia coli. Recombinant LmPDI (lane 1) and MAPKK (lane 2) were synthesized in BL21, purified by affinity chromatography over Ni-nitrilotriacetic acid resin, and analyzed on 14% sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel, followed by Coomassie blue staining. MW, molecular mass markers (kDa).Lymphoproliferative responses (Fig. ​(Fig.2)2) and gamma interferon (IFN-γ) and interleukin-10 (IL-10) production (Fig. ​(Fig.3)3) induced by recombinant LmPDI and MAPKK (10 μg/ml) were characterized in vitro using peripheral blood mononuclear cells (PBMC) from two groups. The first group consisted of 18 hCL patients (age range, 17 to 42 years; mean ± standard deviation [SD], 28 ± 10.9 years) living in an area of L. major infection endemicity (the governorate of Sidi Bouzid, Tunisia). Diagnosis of leishmaniasis was based on the presence of specific scars, leishmanin skin test positivity, and a positive lymphoproliferative response to soluble Leishmania antigen (SLA). The second group consisted of 12 healthy individuals (negative controls) living outside areas of endemicity and having no history of CL. All healed patients showed positive proliferation in response to SLA, with stimulation indices (SI) ranging from 3.04 to 207.76 (mean ± SD, 56.83 ± 58.03) (Fig. ​(Fig.2).2). While no proliferation was observed with LmPDI, MAPKK induced significant PBMC proliferation levels (SI > 2.5) in two of eight hCL individuals. No proliferative responses against SLA and the two recombinant proteins were observed in PBMC from healthy individuals (SI < 2.5) (Fig. ​(Fig.2).2). Polymyxin B treatment of PBMC cultures stimulated with recombinant proteins had very little or no effect on proliferative responses, showing that observed stimulation could not be due to lipopolysaccharide contamination (data not shown).Open in a separate windowFIG. 2.Lymphoproliferative response induced by MAPKK or LmPDI. Levels of lymphocyte proliferation in response to either SLA (10 μg/ml) or recombinant proteins MAPKK (10 μg/ml) and LmPDI (5 μg/ml) incubated for 5 days at 37°C and 5% CO₂ are expressed as SI. The cutoff value (SI = 2.5) is indicated by a horizontal bar.Open in a separate windowFIG. 3.Immune response induced by MAPKK or LmPDI: IFN-γ (A) and IL-10 (B) induction. PBMC from hCL or healthy subjects were stimulated with either SLA (10 μg/ml) or recombinant proteins MAPKK (10 μg/ml) and LmPDI (5 μg/ml). Supernatants were harvested at 48 h and 72 h and were used for quantification of IL-10 and IFN-γ. The dashes indicate the mean cytokine values obtained for the different groups of individuals. NS, nonstimulated cultures.IFN-γ and IL-10 levels in culture supernatants of PBMC stimulated with either SLA (10 μg/ml), MAPKK (10 μg/ml), or LmPDI (5 μg/ml) were determined using an enzyme-linked immunosorbent assay (ELISA) (Fig. 3A and B). As expected, SLA induced significantly high levels of IFN-γ but no IL-10 in hCL individuals (mean IFN-γ production levels ± SD of 1,695 ± 1,983 pg/ml and 39.4 ± 49.7 pg/ml, respectively; P = 0.03) (Fig. 3A and B). Interestingly, MAPKK induced high IFN-γ and IL-10 levels in three of eight hCL individuals tested (mean IFN-γ production levels ± SD of 559 ± 642.5 pg/ml and 26.29 ± 25.33 pg/ml and mean IL-10 production levels ± SD of 2,486 ± 3,394 pg/ml and 316.3 ± 174.8 pg/ml in hCL and healthy individuals, respectively) (Fig. 3A and B). Interestingly, the difference in production between the two groups was highly significant only for IFN-γ (P = 0.007) (Fig. ​(Fig.3A).3A). However, no IFN-γ or IL-10 productions were observed after stimulation of PBMC with LmPDI (Fig. 3A and B). A positive correlation was found between IFN-γ and IL-10 levels (Spearman rank correlation r = 0.594) and between PBMC proliferation and IFN-γ or IL-10 production (Spearman rank correlation r values of 0.6 [P = 0.03] and 0.583 [P = 0.036] for IFN-γ and IL-10, respectively) in response to MAPKK.These results show that MAPKK could constitute a potential vaccine candidate. It is well established that IFN-γ is a key cytokine in resistance against CL and is implicated in parasite killing by activated macrophages (14). However, IL-10 is the main downregulating cytokine of the Th1 immune response and exhibits macrophage-deactivating properties (5, 7). Interestingly, the MAPKK IL-10-inducing capacity was not sufficient for suppression of significant proliferation and high, significant levels of IFN-γ in hCL patients. The IL-10 production by PBMC from hCL as well as healthy individuals might be due to the high level of conservation of MAPKK proteins in eukaryotic species. Although MAPKK stimulates high IL-10 levels, it could constitute a potential vaccine candidate since it was recently reported that the human immune response to crude and defined Leishmania antigens generated during immunization can differ from that induced by natural infection (1). Similarly, MAPKK used as a vaccine might induce a dominant Th1 response compatible with protection.For the second part of the study, we chose to analyze humoral responses to LmPDI and MAPKK in MVL patients rather than CL patients for the following reasons: (i) human sera from CL patients generally show low-level reactivity against Leishmania antigens, compared to sera from MVL patients; (ii) diagnosis of MVL is more problematic than that of CL; and (iii) LmPDI and MAPKK are highly conserved with their Leishmania infantum homologue, with 92% and 97% identities, respectively.The reactivities of MVL patients to LmPDI (10 μg/ml), MAPKK (5 μg/ml), and SLA (2 μg/ml) were assayed by ELISAs with two groups. The first group consisted of 12 MVL children (age range, 1 to 5 years; mean age ± SD, 35 ± 21 months) living in the governorate of Kairouan (a region of MVL endemicity), blood sampled before treatment. Diagnosis of MVL was established on clinical criteria (fever, anemia, splenomegaly, and weight loss) and on demonstration of Leishmania parasites in Giemsa-stained bone marrow smears and/or culture in biphasic Nicolle-Novy-McNeal medium. The second group consisted of 13 healthy volunteers living outside areas of endemicity as negative controls (Fig. ​(Fig.4).4). The cutoff value of reactivity with each antigen was defined as the mean optical density (OD) + 2 SDs obtained with healthy individuals, and these values were equal to 0.18, 0.1, and 0.14 for SLA, MAPKK, and LmPDI, respectively. These cutoff values allowed us to identify positive and negative sera and consequently to estimate the performance parameters of each ELISA (Table ​(Table1).1). ELISAs based on SLA and MAPKK had the best sensitivities, with 100% and 91.7%, respectively. Interestingly, the best specificities and positive predictive values were obtained with MAPKK (100% for MAPKK versus 92.3% for SLA). However, SLA gave the best results for negative predictive values (100% versus 92.9% for MAPKK). The ELISA using LmPDI showed only 66.7% sensitivity, with a specificity of 100%. For all proteins, significant differences in measured OD were observed between MVL patients and healthy controls, with P values of <0.05. Taken together, these results indicate that only MAPKK constitutes a major target of humoral response during MVL.Open in a separate windowFIG. 4.ELISA reactivity of sera from patients with MVL (P) and healthy controls (C) with SLA (2 μg/ml) and recombinant Leishmania proteins MAPKK (5 μg/ml) and LmPDI (10 μg/ml). Bars show the cutoff value for each ELISA, which is defined as the mean OD + 2 SDs for the values obtained with sera from healthy controls. The significance of differences between P and C groups was evaluated by the Mann-Whitney test. P values of <0.05 were considered significant. Arrows indicate mean OD values.

TABLE 1.

Sensitivities and specificities of ELISAs using crude SLAs and recombinant Leishmania proteins MAPKK and LmPDI^a

Sensitivity and Specificity of a Rapid Immunochromatographic Test for Diagnosis of Visceral Leishmaniasis

 A rapid immunochromatographic dipstick test has become available for the qualitative detection of total anti-Leishmania immunoglobulins using the recombinant K39 antigen. To evaluate the test, 96 serum specimens from patients with a variety of tropical infections were tested. Fourteen of the specimens derived from patients with parasitologically confirmed kala-azar, and all were strongly positive for antibodies to Leishmania donovani complex using the immunofluorescence test. Although all 82 samples negative by the immunofluorescence test were confirmed as negative by the dipstick assay, only 10 (71.4%) of the 14 positive samples were reactive. These results indicate that the test in its current form lacks sufficient sensitivity to be recommended as a screening tool, but it might be useful for indicating further diagnostic procedures in a clinical setting.     

Identification of Potentially Diagnostic Leishmania braziliensis Antigens in Human Cutaneous Leishmaniasis by Immunoblot Analysis

The antibody response in patients with American cutaneous leishmaniasis was analyzed by immunoblotting with soluble and insoluble antigens of Leishmania braziliensis. The recognition of the 27- and/or 30-kDa soluble antigens was considered relevant for the diagnosis of cutaneous leishmaniasis. Immunoblotting was found to be significantly more sensitive and specific than indirect immunofluorescence and enzyme-linked immunosorbent assay.     

Killed but Metabolically Active Leishmania infantum as a Novel Whole-Cell Vaccine for Visceral Leishmaniasis

There are currently no effective vaccines for visceral leishmaniasis, the second most deadly parasitic infection in the world. Here, we describe a novel whole-cell vaccine approach using Leishmania infantum chagasi promastigotes treated with the psoralen compound amotosalen (S-59) and low doses of UV A radiation. This treatment generates permanent, covalent DNA cross-links within parasites and results in Leishmania organisms termed killed but metabolically active (KBMA). In this report, we characterize the in vitro growth characteristics of both KBMA L. major and KBMA L. infantum chagasi. Concentrations of S-59 that generate optimally attenuated parasites were identified. Like live L. infantum chagasi, KBMA L. infantum chagasi parasites were able to initially enter liver cells in vivo after intravenous infection. However, whereas live L. infantum chagasi infection leads to hepatosplenomegaly in mice after 6 months, KBMA L. infantum chagasi parasites were undetectable in the organs of mice at this time point. In vitro, KBMA L. infantum chagasi retained the ability to enter macrophages and induce nitric oxide production. These characteristics of KBMA L. infantum chagasi correlated with the ability to prophylactically protect mice via subcutaneous vaccination at levels similar to vaccination with live, virulent organisms. Splenocytes from mice vaccinated with either live L. infantum chagasi or KBMA L. infantum chagasi displayed similar cytokine patterns in vitro. These results suggest that KBMA technology is a potentially safe and effective novel vaccine strategy against the intracellular protozoan L. infantum chagasi. This approach may represent a new method for whole-cell vaccination against other complex intracellular pathogens.     

Prokaryotic Expression and Antigenic Characterization of Three Recombinant Leishmania Antigens for Serological Diagnosis of Canine Leishmaniasis

Three recombinant antigens of Leishmania chagasi (= L. infantum) were expressed in prokaryotic systems and evaluated (using a panel of dog sera characterized by parasitological and serological immunofluorescent antibody test [IFAT] techniques) as diagnostic markers of infection. The whole open reading frame encoding K9, the gene fragment encoding the repetitive sequence of K26, and the 3′-terminal gene fragment encoding a single 39-amino-acid subunit of the kinesin-related protein K39 (K39sub) were amplified from L. infantum DNA and cloned into a pGEX-2T expression vector in frame with glutathione S-transferase (GST). The sensitivity and specificity of enzyme-linked immunosorbent assays (ELISAs) using K26 as an antigen (evaluated with sera from 20 parasitologically positive and 20 parasitologically negative dogs) were both 100% (95% confidence interval [CI] = 83.2 to 100). When K9 and K39sub were used, sensitivity was 95% (95% CI = 75.1 to 99.9) and specificity was 100% (95% CI = 83.2 to 100). Using 182 field sera, a good agreement was found between the recombinant K26 ELISA and IFAT (K = 0.92; 95% CI = 0.86 to 0.98) results and between the K9 and K39sub ELISA (used in parallel) and IFAT (K = 0.87; 95% CI = 0.80 to 0.95) results. The results demonstrate that each antigen carries immunodominant epitopes and that their combination may further increase the sensitivity of currently available serological tests.     

Real-Time PCR as a New Tool for Quantifying Leishmania infantum in Liver in Infected Mice

The parasitic loads of mouse livers experimentally infected with Leishmania infantum were determined using a double real-time quantitative PCR test targeted to the parasite DNA polymerase gene and to the mouse brain-derived neutrophic factor gene. The Leishmania DNA copy number was normalized to the number of mouse gene copies in order to quantify the former independently of liver weight. The correlation coefficient with the microtitration method was 0.66. This PCR assay can be considered for experimental pharmaceutical studies.     

Sensitivity and Specificity of In Situ Hybridization for Diagnosis of Cutaneous Infection by Leishmania infantum in Dogs

An accurate diagnosis of infection by Leishmania infantum in dogs is fundamental for the control of zoonotic visceral leishmaniasis (VL). Histopathology (HP) and immunohistochemistry (IHC) are frequently used for the histological diagnosis of L. infantum in dogs but have shown limited accuracy. To improve the sensitivity and specificity of the histological diagnosis of VL, we evaluated automated in situ hybridization (ISH) using a generic probe for Leishmania and a specific probe for L. infantum in surgical skin biopsy specimens of dogs. The ISH results were compared with those of HP and IHC, using parasitological culture as the reference standard. Skin samples from 51 dogs with cutaneous L. infantum infection and 51 noninfected dogs were randomly selected from samples of dogs from various cities in Brazil where canine VL is endemic. These samples were processed for parasitological culture, HP, IHC, and ISH using both probes. The sensitivities of ISH using the specific probe, ISH using the generic probe, IHC, and HP were, respectively, 74.5%, 70.6%, 69.5%, and 57.6%. The specificity of both ISH probes tested was 100%, and there was no cross-hybridization of the generic and specific probes with selected pathogenic fungi and protozoa. The specific probe discriminated L. infantum from the other species of Leishmania that infect dogs in the New World. ISH is highly sensitive and specific for the diagnosis of L. infantum in histologic samples of skin from infected dogs and can be used on routine biopsy material to make a diagnosis of leishmaniasis.     

Evaluation of Two Novel Rapid rKE16 Antigen-Based Tests for Diagnosis of Visceral Leishmaniasis in India

We compared the two formats of rKE16 antigen-based rapid tests, a flowthrough test (KEFT) and a lateral flow test (KELF), with the rK39 rapid test for the diagnosis of visceral leishmaniasis. Sensitivities with KEFT (99%, 198/200) and rK39 (99.5%, 199/200) were comparable and higher than that with KELF (95.5%, 191/200). In the control groups comprising subjects with diseases from areas of nonendemicity or endemicity and subjects with different diseases, the specificities were comparable for all three rapid tests, except that specificity was higher with KELF in the controls from areas of endemicity.     

IFN-γ and Delayed-Type Hypersensitivity are Associated with Cutaneous Leishmaniasis in Vervet Monkeys following Secondary Rechallenge with Leishmania major

IFN-γ levels and delayed-type hypersensitivity (DIM) responses were evaluated in vervet monkeys, following secondary infection with leishmania major (L. major). The animals had previously been vaccinated with leishmanial antigen, exposed to a primary infection and allowed to self-cure. Supernatants of peripheral blood mononuclear cell (PBMC) cultures, stimulated with either L. major antigen or Concanavalin A (Con A), were examined for the presence of IFN-γ in a double sandwich enzyme-linked immunosorbent ASSAY (ELISA). Significant levels of IFN-γ were detected during active disease and following Self-cure in both antigen and Con A supernatants Higher levels of IFN-y were, however, present during active disease as compared with after self-cure. Positive and strong DTH responses were elicited in all experimental animals, following intradermal injection of fixed promastigotes (5×10⁷/animal) before rechallenge, during active infection and following self-cure. Again, strongest DTH responses were obtained during active infection as compared with the other sampling point
There was a correlation between IFN-γ levels and DTH responses. It was concluded that IFN-γ secretion and positive DTH responses are associated with secondary L. major infection and represent specific immunological correlates of protection in this disease model.     

Evaluation of a Rapid Immunochromatographic Test for Serodiagnosis of Visceral Leishmaniasis

The purpose of this study was to compare the performance of a rapid immunochromatographic dipstick test for the qualitative detection of circulating antibodies to the leishmanial recombinant antigen K39 with that of a classical immunofluorescent antibody test for serodiagnosis of visceral leishmaniasis. Sera from 143 Italian subjects, including 69 patients with clinically suspected visceral leishmaniasis, 23 patients with hypergammaglobulinemia and 51 healthy controls, were tested. The immunochromatographic test was performed according to the manufacturer's instructions, using antigen-impregnated nitrocellulose paper strips. The immunofluorescent antibody test was performed according to an established method, using promastigotes of Leishmania infantum zymodeme Montpellier 1 as antigen. In 11 patients, diagnosis of active Leishmania infection was established by microscopic examination of biopsy samples and/or clinical response to meglumine antimoniate. Results of the two tests correlated for all but two sera examined. In two patients, one with proven infectious mononucleosis and one with bacterial pneumonia, the immunofluorescent antibody test was positive and the dipstick test was negative. In the restricted sample of patients in whom a definitive diagnosis was established, the immunochromatographic test was positive in 11 of 11 patients with confirmed Leishmania infection and negative in 103 of 103 subjects who either had other documented diseases or were healthy controls, showing 100% sensitivity and 100% specificity. Electronic Publication     

Molecular Tools for Diagnosis of Visceral Leishmaniasis: Systematic Review and Meta-Analysis of Diagnostic Test Accuracy

Molecular methods have been proposed as highly sensitive tools for the detection of Leishmania parasites in visceral leishmaniasis (VL) patients. Here, we evaluate the diagnostic accuracy of these tools in a meta-analysis of the published literature. The selection criteria were original studies that evaluate the sensitivities and specificities of molecular tests for diagnosis of VL, adequate classification of study participants, and the absolute numbers of true positives and negatives derivable from the data presented. Forty studies met the selection criteria, including PCR, real-time PCR, nucleic acid sequence-based amplification (NASBA), and loop-mediated isothermal amplification (LAMP). The sensitivities of the individual studies ranged from 29 to 100%, and the specificities ranged from 25 to 100%. The pooled sensitivity of PCR in whole blood was 93.1% (95% confidence interval [CI], 90.0 to 95.2), and the specificity was 95.6% (95% CI, 87.0 to 98.6). The specificity was significantly lower in consecutive studies, at 63.3% (95% CI, 53.9 to 71.8), due either to true-positive patients not being identified by parasitological methods or to the number of asymptomatic carriers in areas of endemicity. PCR for patients with HIV-VL coinfection showed high diagnostic accuracy in buffy coat and bone marrow, ranging from 93.1 to 96.9%. Molecular tools are highly sensitive assays for Leishmania detection and may contribute as an additional test in the algorithm, together with a clear clinical case definition. We observed wide variety in reference standards and study designs and now recommend consecutively designed studies.     

Development of the Korean Standardized Antimicrobial Administration Ratio as a Tool for Benchmarking Antimicrobial Use in Each Hospital

BackgroundThe Korea National Antimicrobial Use Analysis System (KONAS), a benchmarking system for antimicrobial use in hospitals, provides Korean Standardized Antimicrobial Administration Ratio (K-SAAR) for benchmarking. This article describes K-SAAR predictive models to enhance the understanding of K-SAAR, an important benchmarking strategy for antimicrobial usage in KONAS.MethodsWe obtained medical insurance claims data for all hospitalized patients aged ≥ 28 days in all secondary and tertiary care hospitals in South Korea (n = 347) from January 2019 to December 2019 from the Health Insurance Review & Assessment Service. Modeling was performed to derive a prediction value for antimicrobial use in each institution, which corresponded to the denominator value for calculating K-SAAR. The prediction values of antimicrobial use were modeled separately for each category, for all inpatients and adult patients (aged ≥ 15 years), using stepwise negative binomial regression.ResultsThe final models for each antimicrobial category were adjusted for different significant risk factors. In the K-SAAR models of all aged patients as well as adult patients, most antimicrobial categories included the number of hospital beds and the number of operations as significant factors, while some antimicrobial categories included mean age for inpatients, hospital type, and the number of patients transferred from other hospitals as significant factors.ConclusionWe developed a model to predict antimicrobial use rates in Korean hospitals, and the model was used as the denominator of the K-SAAR.     

Leishmania major-Like Antigen for Specific and Sensitive Serodiagnosis of Human and Canine Visceral Leishmaniasis

An antigen (LMS) prepared from Leishmania major-like promastigotes was used in an enzyme-linked immunosorbent assay (ELISA) for the diagnosis of human and dog visceral leishmaniasis. The results were compared with those from the indirect immunofluorescent antibody test (IFAT). A total of 1,822 canine sera were tested, including sera from dogs with visceral leishmaniasis, transmissible venereal tumors, ehrlichiosis, rickettsiosis, or Chagas' disease and sera from healthy dogs. The antigen was also tested with 227 samples of human sera, including sera from patients with visceral, cutaneous, or diffuse cutaneous leishmaniasis and from noninfected individuals, as well as sera from patients with Chagas' disease, toxoplasmosis, rickettsiosis, hepatitis B, schistosomiasis, ascaridiasis, malaria, rheumatoid factor, leprosy and rheumatoid factor, tuberculosis, or leprosy. All dogs and all human patients had a clinical and/or serological and/or parasitological diagnosis. For detecting antibodies in sera from dogs with leishmaniasis, the antigen showed a sensitivity of 98%, specificity of 95%, and concordance of 93% and when used for detecting antibodies in human sera presented a sensitivity of 92%, specificity of 100%, and concordance of 92%. Comparison between ELISA and IFAT demonstrated that ELISA using the LMS antigen yielded more reliable results than IFAT. The LMS antigen displayed no cross-reactivity with sera from patients or dogs that had any of the other diseases tested.     

Molecular Characterisation of Staphylococcus aureus Using spa Typing as a Diagnostic Tool in Haryana,India

Background: Staphylococcus aureus is one of the top six most common etiologic agents of nosocomial, community and livestock acquired bacterial infections. These infections although initially were described as a major problem in hospitals have now also become a serious threat in community not only in India but also worldwide. Its prevalence varies depending on the health-care setting, country or a particular region. Thus to better understand the epidemiology of methicillin-resistant S. aureus (MRSA) in a particular geographical location, it is important to study the variations in the population using molecular tools. Methods: This prospective study was carried out in the Department of Microbiology of Shree Guru Gobind Singh Tricentenary (SGT) Medical College. Staphylococcal protein A (spa) typing was done on 250 S. aureus isolates obtained from various clinical specimens including pus, wound swabs, urine, catheters, blood and cerebrosspinal fluid from both indoor and outdoor patients of SGT Hospital, Budhera, Gurgaon. Results: The selected region of the spa gene of all 250 isolates which includes 87 MRSA and 163 methicillin-susceptible S. aureus were amplified. The spa gene was detected in 248 out of 250 isolates (99.2%), whereas in 2 isolates (0.8%), it remained undetected and referred as non-typable isolates. The 248 S. aureus isolates were typed into 39 spa types, which clustered into six different spa clonal clusters and eight singletons. Conclusion: High diversity observed within S. aureus isolates indicated that many different strains circulate in the study region or in the hospital. The results would contribute in the understanding of epidemiology related to S. aureus spread and prevention.     

Evaluation of a microculture method for isolation of Leishmania parasites from cutaneous lesions of patients in Peru

Traditional culture of Leishmania spp. is labor intensive and has poor sensitivity. We evaluated a microculture method for the diagnosis of cutaneous leishmaniasis in consecutive patients presenting to the Leishmaniasis Clinic at the Instituto de Medicina Tropical Alexander von Humboldt, Peru, for evaluation of skin lesions. Lesion aspirates were cultured in duplicate and parallel in traditional culture tubes containing modified Novy-MacNeal-Nicolle (NNN) medium or Roswell Park Memorial Institute medium 1640 with 10% fetal bovine serum (10% RPMI) and in 70-microl capillary tubes containing a mixture of lesion aspirate and 10% RPMI. For sensitivity analysis, the consensus standard was considered to be a positive result in any two of the following four tests: Giemsa-stained lesion smear, culture, kinetoplast DNA PCR, or leishmanin skin test. The outcome measures were sensitivity and time to culture positivity. Forty-five patients with 62 skin lesions were enrolled in the study, of which 53 lesions fulfilled the consensus criteria for a final diagnosis of cutaneous leishmaniasis. Of these 53 lesions, 39 were culture positive: 38 in capillary tubes, 29 in traditional culture tubes with modified NNN medium, and 19 in traditional culture tubes with 10% RPMI medium. The sensitivity of microculture was 71.7%, versus 54.7% for traditional culture with NNN (P, 0.038) and 35.8% with 10% RPMI (P, <0.001). The mean times to culture positivity were 4.2 days by microculture, 5.2 days in NNN, and 6 days in 10% RPMI (P, 0.009). We have demonstrated that microculture is a more sensitive and time-efficient means of isolating Leishmania parasites from cutaneous lesions than traditional culture.