Diagnostic accuracy of automation and non-automation techniques for identifying Burkholderia pseudomallei: A systematic review and meta-analysis.

BACKGROUND: Burkholderia pseudomallei, a Gram-negative pathogen, causes melioidosis. Although various clinical laboratory identification methods exist, culture-based techniques lack comprehensive evaluation. Thus, this systematic review and meta-analysis aimed to assess the diagnostic accuracy of culture-based automation and non-automation methods. METHODS: Data were collected via PubMed/MEDLINE, EMBASE, and Scopus using specific search strategies. Selected studies underwent bias assessment using QUADAS-2. Sensitivity and specificity were computed, generating pooled estimates. Heterogeneity was assessed using I2 statistics. RESULTS: The review encompassed 20 studies with 2988 B. pseudomallei samples and 753 non-B. pseudomallei samples. Automation-based methods, particularly with updating databases, exhibited high pooled sensitivity (82.79%; 95% CI 64.44-95.85%) and specificity (99.94%; 95% CI 98.93-100.00%). Subgroup analysis highlighted superior sensitivity for updating-database automation (96.42%, 95% CI 90.01-99.87%) compared to non-updating (3.31%, 95% CI 0.00-10.28%), while specificity remained high at 99.94% (95% CI 98.93-100%). Non-automation methods displayed varying sensitivity and specificity. In-house latex agglutination demonstrated the highest sensitivity (100%; 95% CI 98.49-100%), followed by commercial latex agglutination (99.24%; 95% CI 96.64-100%). However, API 20E had the lowest sensitivity (19.42%; 95% CI 12.94-28.10%). Overall, non-automation tools showed sensitivity of 88.34% (95% CI 77.30-96.25%) and specificity of 90.76% (95% CI 78.45-98.57%). CONCLUSION: The study underscores automation's crucial role in accurately identifying B. pseudomallei, supporting evidence-based melioidosis management decisions. Automation technologies, especially those with updating databases, provide reliable and efficient identification.

Stimulation of metacyclogenesis in Leishmania (Mundinia) orientalis for mass production of metacyclic promastigotes.

Leishmania (Mundinia) orientalis is a human pathogen causing leishmaniasis and studies on the properties of metacyclic promastigotes, the parasite's infective stage, are required for a better understanding of its transmission and infection. However, information on cultivation for mass production of L. orientalis metacyclic promastigotes and factors that stimulate their metacyclogenesis is limited. Therefore, the objective of this study was to develop a suitable methodology for generating promastigote cultures containing a high proportion and number of L. orientalis metacyclic promastigotes. Various media, i.e., Schneider's insect medium, Medium 199 and Grace's insect medium, supplemented with various quantities of dithiothreitol, Basal Medium Eagle vitamins, pooled human urine, and fetal bovine serum, were optimized for metacyclogenesis. The results revealed that the optimum culture medium and conditions of those tested were Schneider's insect medium supplemented with 100 µM dithiothreitol, 1% (v/v) Basal Medium Eagle vitamins, 2% (v/v) pooled human urine, and 10% (v/v) fetal bovine serum, pH 5.0 at 26°C. We also demonstrated that L. orientalis metacyclic promastigotes could be purified and enriched by negative selection using peanut lectin. Under these culture conditions, the highest yield of metacyclic promastigotes was obtained with a significantly higher percentage of parasite survival, resistance to complement-mediated lysis, and infection index in THP-1 macrophage cells compared to parasites cultured without media supplements at neutral pH. This is the first report providing a reliable method for mass production of L. orientalis metacyclic promastigotes for in vivo infections and other experimental studies of this emerging parasite in the future.

In vitro anti-Leishmania activity of 8-hydroxyquinoline and its synergistic effect with amphotericin B deoxycholate against Leishmania martiniquensis.

Leishmania (Mundinia) martiniquensis is responsible for visceral leishmaniasis in patients with no known underlying immunodeficiency, and visceral or disseminated cutaneous leishmaniasis in HIV-infected patients. The available anti-Leishmania drugs for treatment have limitations such as high toxicity and variable efficacy. To improve the therapeutic index of anti-Leishmania drugs, the search for a new drug or a new natural compound in combination therapy instead of using monotherapy to reduce drug side effect and have high efficacy is required. In this study, anti-Leishmania activity of 8-hydroxyquinoline (8HQN) and its synergistic effect with amphotericin B (AmB) against L. martiniquensis were evaluated in vitro for the first time. These results showed that 8HQN presented anti-Leishmania activity against L. martiniquensis with IC50 1.60 ± 0.28 and 1.56 ± 0.02 µg/mL for promastigotes and intracellular amastigotes, respectively. The selectivity index (SI) value of 8HQN was 79.84 for promastigotes and 82.40 for intracellular amastigotes, which highlight promising results for the use of 8HQN in the treatment of L. martiniquensis-infected host cells. Interestingly, four combinations of 8HQN and AmB provided synergistic effects for intracellular amastigotes and showed no toxic effects to host cells. These results provided information of using a combination therapy in treating this Leishmania species leads to further development of therapy and can be considered as an alternative treatment for leishmaniasis.

Anti-Leishmania activity of artesunate and combination effects with amphotericin B against Leishmania (Mundinia) martiniquensis in vitro.

Leishmaniasis is an emerging disease in several countries over the world, especially in tropical regions. In Thailand, Leishmania (Mundinia) martiniquensis is the most frequent cause of visceral leishmaniasis and disseminated cutaneous leishmaniasis among HIV/AIDs patients. Amphotericin B (AmB) is the only drug currently available for the treatment of leishmaniasis in Thailand, but has some limitations like high renal toxicity and the prolonged hospitalization required for the treatment. Moreover, recurrence of the disease has been reported in several cases, indicating that new drugs or treatment strategies should be improved. In this study, Artesunate (ARS) was determined for anti-Leishmania activity against L. martiniquensis in promastigotes and amastigotes. In addition, the combination effects of ARS and AmB against intracellular amastigotes on THP-1 derived macrophages were also investigated for the first time. The result showed that L. martiniquensis was susceptible to ARS in both stages of the parasite. ARS was effective against intracellular amastigotes and safe to macrophage host cells, showing a SI value of 1,065. Furthermore, combination effects of ARS and AmB showed five synergistic combinations with a combination index (CI) value less than 1.0 (0.28-0.92) for intracellular amastigotes ranging from slight synergism to strong synergism. The strong synergistic combination had the highest dose reduction index (DRI), approximately a 9.7-fold reduction in AmB used. None of the treatments in combination had noticeable toxicity to THP-1 derived macrophages in the concentration range examined. The data provided in this study lead to further study in vivo and to develop a novel formulation of drug combinations to improve the outcome of leishmaniasis treatment.

Prevalence and characteristics of malaria co-infection among individuals with visceral leishmaniasis in Africa and Asia: a systematic review and meta-analysis.

BACKGROUND: Malaria and visceral leishmaniasis (VL) co-infection can occur due to the overlapping geographical distributions of these diseases; however, only limited data of this co-infection have been reported and reviewed. This study aimed to explore the pooled prevalence and characteristics of this co-infection using a systematic review approach. METHODS: The PubMed, Web of Science and Scopus databases were searched for relevant studies. The quality of these studies was assessed in accordance with strengthening the reporting of observational studies in epidemiology (STROBE) guidelines. The numbers of individuals co-infected with Plasmodium and VL and the total numbers of individuals with VL were used to estimate the pooled prevalence using random-effects models. Differences in age, sex and the presence of anemia and malnutrition on admission were compared between co-infected individuals and individuals with VL using a random-effects model; the results are presented as odds ratios (ORs) and 95% confidence intervals (CIs). Heterogeneity among the included studies was assessed and quantified using Cochrane Q and I2 statistics. RESULTS: Of the 3075 studies identified, 12 met the eligibility criteria and were included in this systematic review. The pooled prevalence of Plasmodium infection among the 6453 individuals with VL was 13%, with substantial heterogeneity of the data (95% CI 7-18%, I2 97.9%). Subgroup analysis demonstrated that the highest prevalence of co-infection occurred in African countries, whereas the lowest prevalence occurred in Asian countries. Patients aged < 5 years had higher odds of having co-infection than having VL (co-infection, n = 202; VL, n = 410) (OR 1.66, 95% CI 1.37-2.01, I2 0%; P < 0.0001), whereas patients aged 20-29 years had lower odds of having co-infection than having VL (co-infection, n = 170; VL, n = 699) (OR 0.75, 95% CI 0.60-0.93, I2 18%; P = 0.01). Male patients had equivalent odds of having co-infection and having VL (co-infection, n = 525; VL, n = 2232) (OR 0.92, 95% CI 0.078-1.08, I2 0%; P = 0.29). Patients with co-infection had lower odds of having anemia at admission than those with VL (co-infection, n = 902; VL, n = 2939) (OR 0.64, 95% CI 0.44-0.93, I2 0%; P = 0.02). No difference in malnutrition at admission was found in the meta-analysis. CONCLUSIONS: The prevalence of malaria co-infection among individuals with VL was heterogeneous and ranged from 7 to 18%, depending on geographical area. Age and anemia at admission were associated with co-infection status. Further longitudinal studies are needed to determine if co-infection with malaria has an impact on the severity of VL.

Experimental infection of Leishmania (Mundinia) martiniquensis in BALB/c mice and Syrian golden hamsters.

Our objective was to investigate clinical progression, presence of parasites and DNAs, parasite loads, and histological alterations in BALB/c mice and Syrian golden hamsters after intraperitoneal inoculation with Leishmania (Mundinia) martiniquensis promastigotes with a goal to choosing an appropriate animal model for visceral leishmaniasis. Infections were monitored for 16 weeks. Infected BALB/c mice were asymptomatic during the infection course. Parasite DNAs were detected in the liver at week 8 of infection, followed by clearance in most animals at week 16; whereas in the spleen, parasite DNAs were detected until week 16. These results are correlated to those obtained measuring parasite loads in both organs. No parasite DNA and no alteration in the bone marrow were observed indicating that no dissemination occurred. These results suggest the control of visceralization of L. martiniquensis by BALB/c mice. In hamsters, weight loss, cachexia, and fatigue were observed after week 11. Leishmania martiniquensis parasites were observed in tissue smears of the liver, spleen, and bone marrow by week 16. Parasite loads correlated with those from the presence of parasites and DNAs in the examined tissues. Alterations in the liver with nuclear destruction and cytoplasmic degeneration of infected hepatocytes, presence of inflammatory infiltrates, necrosis of hepatocytes, and changes in splenic architecture and reduction and deformation of white pulp in the spleen were noted. These results indicate a chronic form of visceral leishmaniasis indicating that the hamster is a suitable animal model for the study of pathological features of chronic visceral leishmaniasis caused by L. martiniquensis.

Antileishmanial Activity and Synergistic Effects of Amphotericin B Deoxycholate with Allicin and Andrographolide against Leishmania martiniquensis In Vitro.

Leishmania (Mundinia) martiniquensis is a causative agent of visceral leishmaniasis, but in HIV-infected patients both visceral and disseminated cutaneous leishmaniasis are presented. Recurrence of the disease after treatment has been reported in some cases indicating that improved chemotherapy is required. In this study, the susceptibility of L. martiniquensis to Amphotericin B deoxycholate (AmB), allicin, and andrographolide was evaluated and the synergistic effects of allicin or andrographolide combined with AmB against L. martiniquensis intracellular amastigotes in mouse peritoneal exudate macrophages (PEMs) were investigated in vitro for the first time. The results showed that L. martiniquensis was highly susceptible to AmB as expected, but allicin and andrographolide had selectivity index (SI) values greater than 10, indicating promise in both compounds for treatment of host cells infected with L. martiniquensis. Four AmB/allicin combinations presented combination index (CI) values less than 1 (0.58-0.68) for intracellular amastigotes indicating synergistic effects. The combination with the highest dose reduction index (DRI) allowed an approximately four-fold reduction of AmB use in that combination. No synergistic effects were observed in AmB/andrographolide combinations. The data provided in this study leads for further study to develop novel therapeutic agents and improve the treatment outcome for leishmaniasis caused by this Leishmania species.

Development of Leishmania orientalis in the sand fly Lutzomyia longipalpis (Diptera: Psychodidae) and the biting midge Culicoides soronensis (Diptera: Ceratopogonidae).

Leishmania (Mundinia) orientalis is a newly described species causing human leishmaniasis in Thailand whose natural vector is unknown. L. orientalis infections in sand flies and/or biting midges under laboratory conditions have not been previously investigated. In this study, the development of L. orientalis in two experimental vectors, Lutzomyia longipalpis sand flies and Culicoides sonorensis biting midges was investigated for the first time using light microscopy, scanning electron microscopy, and histological examination. The results showed that L. orientalis was unable to establish infection in Lu. longipalpis. No parasites were found in the sand fly gut 4 days post-infected blood meal (PIBM). In contrast, the parasite successfully established infection in C. sonorensis. The parasites differentiated from amastigotes to procyclic promastigotes in the abdominal midgut (AMG) on day 1 PIBM. On day 2 PIBM, nectomonad promastigotes were observed in the AMG and migrated to the thoracic midgut (TMG). Leptomonad promastigotes appeared at the TMG on day 3 PIBM. Clusters of leptomonad promastigotes and metacyclic promastigotes colonized around the stomodeal valve with the accumulation of a promastigote secretory gel-like material from day 3 PIBM onwards. Haptomonad-like promastigotes were observed from day 5 PIBM, and the proportion of metacyclic promastigotes reached 23% on day 7 PIBM. The results suggest that biting midges or other sand fly genera or species might be vectors of L. orientalis.

Axenic amastigote cultivation and in vitro development of Leishmania orientalis.

Leishmania (Mundinia) orientalis is a recently described new species that causes leishmaniasis in Thailand. To facilitate characterization of this new species, an in vitro culture system to generate L. orientalis axenic amastigotes was developed. In vitro culture conditions of the axenic culture-derived amastigotes were optimized by manipulation of temperature and pH. Four criteria were used to evaluate the resulting L. orientalis axenic amastigotes, i.e., morphology, zymographic analysis of nucleases, cyclic transformation, and infectivity to the human monocytic cell line (THP-1) cells. Results revealed that the best culture condition for L. orientalis axenic amastigotes was Grace's insect medium supplemented with FCS 20%, 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 5.5 at 35 °C. For promastigotes, the condition was M199 medium, 10% FCS supplemented with 2% human urine, 1% BME vitamins, and 25 µg/ml gentamicin sulfate, pH 6.8 at 26 °C. Morphological characterization revealed six main stages of the parasites including amastigotes, procyclic promastigotes, nectomonad promastigotes, leptomonad promastigotes, metacyclic promastigotes, and paramastigotes. Also, changes in morphology during the cycle were accompanied by changes in zymographic profiles of nucleases. The developmental cycle of L. orientalis in vitro was complete in 12 days using both culture systems. The infectivity to THP-1 macrophages and intracellular growth of the axenic amastigotes was similar to that of THP-1 derived intracellular amastigotes. These results confirmed the successful axenic cultivation of L. orientalis amastigotes. The axenic amastigotes and promastigotes can be used for further study on infection in permissive vectors and animals.

Leishmania (Mundinia) orientalis n. sp. (Trypanosomatidae), a parasite from Thailand responsible for localised cutaneous leishmaniasis.

BACKGROUND: Leishmaniasis is an emerging disease in Thailand with an unknown incidence or prevalence. Although the number of properly characterized and clinically confirmed cases is about 20, it is suspected that this low number masks a potentially high prevalence, with clinical disease typically manifesting itself against an immunocompromised background, but with a substantial number of subclinical or cured cases of infection. To date leishmaniasis in Thailand has been mainly ascribed to two taxa within the recently erected subgenus Mundinia Shaw, Camargo & Teixeira, 2016, Leishmania (Mundinia) martiniquensis Desbois, Pratlong & Dedet, 2014 and a species that has not been formally described prior to this study. RESULTS: A case of simple cutaneous leishmaniasis was diagnosed in a patient from Nan Province, Thailand. Molecular analysis of parasites derived from a biopsy sample revealed this to be a new species of Leishmania Ross, 1908, which has been named as Leishmania (Mundinia) orientalis Bates & Jariyapan n. sp. A formal description is provided, and this new taxon supercedes some isolates from the invalid taxon "Leishmania siamensis". A summary of all known cases of leishmaniasis with a corrected species identification is provided. CONCLUSIONS: Three species of parasites are now known to cause leishmaniasis is Thailand, L. martiniquensis and L. orientalis n. sp. in the subgenus Mundinia, which contains the type-species Leishmania enriettii Muniz & Medina, 1948, and a single case of Leishmania infantum Nicolle, 1908. This study now enables epidemiological and other investigations into the biology of these unusual parasites to be conducted. It is recommended that the use of the taxonomically invalid name "L. siamensis" should be discontinued.

Identification of salivary gland proteins depleted after blood feeding in the malaria vector Anopheles campestris-like mosquitoes (Diptera: Culicidae).

Malaria sporozoites must invade the salivary glands of mosquitoes for maturation before transmission to vertebrate hosts. The duration of the sporogonic cycle within the mosquitoes ranges from 10 to 21 days depending on the parasite species and temperature. During blood feeding salivary gland proteins are injected into the vertebrate host, along with malaria sporozoites in the case of an infected mosquito. To identify salivary gland proteins depleted after blood feeding of female Anopheles campestris-like, a potential malaria vector of Plasmodium vivax in Thailand, two-dimensional gel electrophoresis and nano-liquid chromatography-mass spectrometry techniques were used. Results showed that 19 major proteins were significantly depleted in three to four day-old mosquitoes fed on a first blood meal. For the mosquitoes fed the second blood meal on day 14 after the first blood meal, 14 major proteins were significantly decreased in amount. The significantly depleted proteins in both groups included apyrase, 5'-nucleotidase/apyrase, D7, D7-related 1, short form D7r1, gSG6, anti-platelet protein, serine/threonine-protein kinase rio3, putative sil1, cyclophilin A, hypothetical protein Phum_PHUM512530, AGAP007618-PA, and two non-significant hit proteins. To our knowledge, this study presents for the first time the salivary gland proteins that are involved in the second blood feeding on the day corresponding to the transmission period of the sporozoites to new mammalian hosts. This information serves as a basis for future work concerning the possible role of these proteins in the parasite transmission and the physiological processes that occur during the blood feeding.

MIDGUT ULTRASTRUCTURE OF FOURTH INSTAR OCHLEROTATUS TOGOI (DIPTERA: CULICIDAE).

The ultrastructure of the midgut of fourth instar Ochlerotatus togoi was investigated by light, scanning and transmission electron microscopy. This study was performed to provide information to help devise future control efforts aimed at the larval stages of this vector of filariasis. The fourth instar midgut was approximately 2 mm in length and consisted of three morphologically distinct cell types: epithelial, regenerative, and endocrine cells. There was a monolayer of epithelial cells on the luminal surface of the midgut, with multiple folds of the plasma membrane where it adjoined the basement membrane. Regenerative cells were scattered throughout the basal portion of the epithelium, along with endocrine cells. No evidence of division or differentiation was seen in any of the cell types. Six layers of the peritrophic matrix were observed in the gut lumen which separated ingested food from the midgut epithelial cells. Cytoplasmic protrusions were seen in many areas of the luminal midgut surface and numerous autophagosomes were seen in the epithelial cells of both early and late fourth instar larvae, suggesting autophagy is involved in the degeneration process of the midgut in preparation for pupation. This study provides a basis for understanding normal Oc. togoi larval midgut development. Further studies are needed to determine the factors that control larval growth and the nutritional state. Such information could be used to reduce adult fecundity and develop biological control mechanisms.

Peritrophic matrix formation and Brugia malayi microfilaria invasion of the midgut of a susceptible vector, Ochlerotatus togoi (Diptera: Culicidae).

The mosquito midgut is the first site that vector-borne pathogens contact during their multiplication, differentiation, or migration from blood meal to other tissues before transmission. After blood feeding, the mosquitoes synthesize a chitinous structure called peritrophic matrix (PM) that envelops the blood meal and separates the food bolus from the midgut epithelium. In this study, a systematic investigation of the PM formation and the interaction of Brugia malayi within the midgut of a susceptible vector, Ochlerotatus togoi, were performed using scanning electron microscopy (SEM). SEM analysis of the midguts dissected at different time points post feeding on a B. malayi-infected blood meal (PIBM) revealed that the PM was formed from 45 min PIBM and gradually thickened and matured during 8-18 h PIBM. The PM degraded from 24 to 72 h PIBM, when digestion was completed. The invasion process of the microfilariae was observed between 3 and 4 h PIBM. In the beginning of the process, only sheathed microfilariae interacted with the internal face of the PM by its anterior part, and then the midgut epithelium before entering the hemocoel, after that they exsheathed. Microfilarial sheaths lying within the hemocoel were observed suggesting that they may serve as a decoy to induce the immune systems of the mosquitoes to respond to the antigens on the sheaths, thereby protecting the exsheathed microfilariae. These initial findings would lead to further study on the proteins, chemicals, and factors in the midgut that are involved in the susceptibility of O. togoi as a vector of filariasis.