Detection of Macrolide-Resistant Streptococcus pneumoniae Genes and Its Clinical Outcomes in a Tertiary Teaching Hospital in Malaysia.

Background: Streptococcus pneumoniae is one of the leading causes of mortality and morbidity worldwide. The dramatic increase in in-vitro resistance of antimicrobial agents, particularly beta-lactams and macrolides, makes pneumococcal infections difficult to treat. The aim of this study was to describe the drug resistance rate, assess the prevalence of macrolide-resistant genes and review the clinical complications of pneumococcal infections among patients presented to Hospital Universiti Sains Malaysia (HUSM), Kelantan, Malaysia. Methods: This is a descriptive cross-sectional study. All S. pneumoniae isolates collected from clinical specimens within a 1-year period were subjected to selected antimicrobial susceptibility testing using E-test strips. Polymerase chain reaction (PCR) analysis was conducted to detect macrolide-resistant determinants. The patient's clinical data were obtained from clinical notes. Results: A total of 113 patients with a positive growth of S. pneumoniae were included in the study. The most common predisposing factors among them were bronchopulmonary diseases (15.9%). The penicillin-resistant rate was 7.1%, with minimal inhibitory concentration (MIC) ranging between 0.012 µg/mL and >32 µg/mL, and the erythromycin-resistant rate was 26.5%, with a MIC range of 0.03 µg/mL-> 256 µg/mL. Most of the erythromycin-resistant isolates were found to have the mef(A) gene (50.4%) and the erm(B) gene (20%); 16.7% had a combination of genes mef(A) and erm(B), and 13.3% had none of the two genes. Community-acquired pneumonia is the predominant type of pneumococcal infection. There was no significant association between the presence of macrolide resistance determinants and mortality (P = 0.837) or complications (P > 0.999 for empyema and cardiac complication; P = 0.135 for subdural abscess). Conclusion: The majority of erythromycin-resistant isolates were found to have the mef(A) gene, followed by the erm(B) gene and a combination of genes mef(A) and erm(B).

Environmentally Benign Water-Soluble Sodium L-2-(1-Imidazolyl) Alkanoic Acids as New Corrosion Inhibitors for Mild Steel in Artificial Seawater.

Three novel natural amino acid-derived sodium L-2-(1-imidazolyl) alkanoic acids (IZSs), namely, sodium 2-(1H-imidazol-1-yl)-4-methylpentanoate (IZS-L), sodium 2-(1H-imidazol-1-yl)-3-phenylpropanoate (IZS-P), and sodium 2-(1H-imidazol-1-yl)-4-(methylthio)butanoate (IZS-M), were investigated as corrosion inhibitors. The IZSs were synthesized following the green chemistry principles, and their structure was characterized using FTIR and NMR techniques. The corrosion study results reveal that a moderate concentration of IZSs (having low solution conductivity) showed potential corrosion inhibition for mild steel in artificial seawater. At longer immersion, IZS-P forms a uniform protective film and exhibits the potential inhibition efficiency of 82.46% at 8.4 mmol L-1. Tafel polarization results reveal that IZS-P and IZS-M act as mixed types with an anodic predominantly corrosion inhibitor. The electrochemical impedance spectroscopy results signify that IZSs inhibit mild steel corrosion through the formation of an inhibitor film on the metal surface, which was further confirmed by the FTIR, SEM, EDX, and XPS studies. DFT result shows that in IZS-P, the benzylic group (-CH2-Ph) has greater electron distribution compared to isobutyl (-CH2CH(CH3)2) in IZS-L and methythioethyl group (-CH2CH2SCH3) which supported the corrosion inhibition performance at longer immersion [IZS-P (82.46%) > IZS-M (67.19%) > IZS-L (24.77%)].

Pediatric brain abscess with fatal outcome caused by hypervirulent Klebsiella pneumoniae, serotype K2-ST65.

Hypervirulent Klebsiella pneumoniae (hvKp) is an emerging pathotype in addition to classical Klebsiella pneumoniae, with its ability to cause life-threatening, community-acquired metastatic infections even in healthy individuals. We presented a case of cerebral abscess preceded by otitis media in a 10-year-old child caused by hvKp. The isolates from blood pus aspirate were later identified as K. pneumoniae capsular serotype K2 and closely related to sequence type (ST65), with multiple hypervirulent genes detected (rmpA, rmpA2, iucA and peg344). She succumbed to death despite surgical drainage and susceptible antibiotic therapy. Clinicians should be cognizant of the rising incidence of hvKp infections in pediatric populations.

Low Seroprevalence of SARS-CoV-2 among Healthcare Workers in Malaysia during the Third COVID-19 Wave: Prospective Study with Literature Survey on Infection Prevention and Control Measures.

Healthcare workers (HCWs) are at greater risk for severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infection. This serology surveillance study aimed to investigate the prevalence of SARS-CoV-2 antibodies among the HCWs who were asymptomatic during the third wave of COVID-19 in Malaysia. HCWs from the Universiti Sains Malaysia (USM) Health Campus were prospectively recruited between August 2020 and March 2021 on a voluntary basis. Data on socio-demographics, possible risk factors and travel history were recorded. Serological diagnoses from serum samples were examined for total antibodies against SARS-CoV-2 using an immunoassay kit. A literature survey was performed on the compliance with infection and prevention control (IPC) practices for COVID-19 among HCWs. The majority of the total 617 HCWs participating in this study were nurses (64.3%, n = 397), followed by health attendants (20.9%, n = 129), medical doctors (9.6%, n = 59) and others (6.3%, n = 39). Of those, 28.2% (n = 174) claimed to have exposure to COVID-19 cases, including history of close contact and casual contact with infected patients. Most importantly, all serum samples were found to be non-reactive to SARS-CoV-2, although nearly half (40.0%, n = 246) of the HCWs had been involved directly in the management of acute respiratory illness cases. A proportion of 12.7% (n = 78) of the HCWs reported having underlying health problems, such as diabetes mellitus, hypertension and hyperlipidemia. Despite the presence of medical and sociological risks associated with SARS-CoV-2 infections, the current study found zero prevalence of antibodies against SARS-CoV-2 among the HCWs of USM. Based on the literature survey, the vast majority of Malaysian HCWs demonstrated good IPC practices during the pandemic (average percentage ranged between 92.2% and 99.8%). High compliance with IPC measures may have led to the low seroprevalence of SARS-CoV-2 among the HCWs.

Putative Pathogenic Genes of Leptospira interrogans and Leptospira weilii Isolated from Patients with Acute Febrile Illness.

Leptospirosis is an important worldwide tropical disease caused by pathogenic Leptospira spp. The determination of virulence genes is important, as it influences patients' clinical manifestations and clinical outcomes. This case report focused on detecting the pathogenic genes of Leptospira in association with the clinical manifestations of patients at the Hospital Universiti Sains Malaysia, Malaysia, who presented with acute febrile illness. Two cases were found and, to the best of our knowledge, these were the first two cases in Malaysia in which patients presented with febrile illness were associated with successful Leptospira isolation from clinical samples. Both clinical isolates were identified by 16S rRNA sequencing as Leptospira weilii and Leptospira interrogans, respectively, and they were classified as pathogenic Leptospira by the presence of different pathogenic genes, based on a polymerase chain reaction (PCR) amplification of targeted genes. This report emphasizes that different infecting Leptospira species and the presence of different virulence factors cause a slight difference in clinical manifestations and laboratory findings of leptospirosis. Genomic sequencing and annotation revealed the detection of classical leptospiral virulence factor genes that were otherwise missed using PCR for detection of Leptospira weilii genome B208.

Seroprevalence of SARS-CoV-2 Antibodies in Africa: A Systematic Review and Meta-Analysis.

A reliable estimate of SARS-CoV-2-specific antibodies is increasingly important to track the spread of infection and define the true burden of the ongoing COVID-19 pandemic. A systematic review and a meta-analysis were conducted with the objective of estimating the seroprevalence of SARS-CoV-2 infection in Africa. A systematic search of the PubMed, Scopus, Web of Science and Google Scholar electronic databases was conducted. Thirty-five eligible studies were included. Using meta-analysis of proportions, the overall seroprevalence of anti-SARS-CoV-2 antibodies was calculated as 16% (95% CI 13.1-18.9%). Based on antibody isotypes, 14.6% (95% CI 12.2-17.1%) and 11.5% (95% CI 8.7-14.2%) were seropositive for SARS-CoV-2 IgG and IgM, respectively, while 6.6% (95% CI 4.9-8.3%) were tested positive for both IgM and IgG. Healthcare workers (16.3%) had higher seroprevalence than the general population (11.7%), blood donors (7.5%) and pregnant women (5.7%). The finding of this systematic review and meta-analysis (SRMA) may not accurately reflect the true seroprevalence status of SARS-CoV-2 infection in Africa, hence, further seroprevalence studies across Africa are required to assess and monitor the growing COVID-19 burden.

In Vitro Anti-Leptospiral Activity of Phyllanthus amarus Extracts and Their Combinations with Antibiotics.

Despite modern medicine, there is an increasing trend for cases of the bacterial infection leptospirosis, and this has led to the exploration of alternative medicines from various sources including plants. The aim of this study was to investigate the in vitro anti-leptospiral activity of Phyllanthus amarus extracts alone and combined with penicillin G, ceftriaxone, and doxycycline. Antimicrobial susceptibility testing was performed using the microdilution broth technique upon methanol extract (ME), aqueous extract (AE), and antibiotics against the Leptospira interrogans serovars Australis, Bataviae, Canicola, and Javanica, to determine minimum inhibitory concentrations (MICs) and minimum bactericidal concentrations (MBCs). The results were analyzed using an ELISA microplate reader combined with microscopic analysis. Synergy testing using a checkerboard assay was performed to determine the fractional inhibitory concentration index values of extracts combined with antibiotics against leptospires. Scanning electron microscopy (SEM) was used to investigate morphological changes of leptospires caused by potential anti-leptospiral agents alone and combined with antibiotics. The MICs and MBCs for P. amarus extracts ranged from 100 to 400 µg/mL for AEs and from 400 to 800 µg/mL for MEs. Penicillin G was the most effective anti-leptospiral drug, with MICs and MBCs ranging from <0.01 to 0.78 and <0.01 to 3.13 µg/mL, respectively, followed by ceftriaxone, with both MICs and MBCs ranging from 0.05 to 0.78 µg/mL, and doxycycline, with MICs and MBCs ranging from 0.39 to 3.13 µg/mL and 12.5 to 25 µg/mL, respectively. Combinations of P. amarus extracts and antibiotics did not show synergistic effects on all tested Leptospira serovars, with some combinations demonstrating antagonistic effects. SEM analysis, however, showed distorted Leptospira surfaces. P. amarus AE performed better anti-leptospiral activity than P. amarus ME. The morphological effects of P. amarus extract alone and its combination with antibiotic on Leptospira cells revealed promising anti-leptospiral properties.

Dataset in characterization of the polymer produced using different method of synthesis polychloromethylstyrene (PCMS) with clay and without clay.

Polychloropolymethylstyrene (PCMS) polymers were synthesized with clay Cloisite and without clay Cloisite and chloromethylstyrene (CMS) combine with styrene (1:1) v/v or known as copolymer and clay Cloisite by the polymerization process. The attenuated total reflection-Fourier transform infrared (ATR-FTIR) spectra of each polymer synthesized are reported. The spectra of IR shows the different value of the wavenumber and intensity for each set of different sample. The spectra can be as a reference for others to use in synthesizing this polymer and clay Cloisite for different type of application.

Leptospiral Culture without 5'-Fluorouracil Revealed Improved Leptospira Isolation from Febrile Patients in North-Eastern Malaysia.

Objectives: Isolation of Leptospira by culture represents a definitive growth and confirmation of the disease, yet it is hampered with its nature of slow growth. With slight modification of culture method, the study aims to isolate and characterize Leptospira spp. from patients with acute febrile illness. Methods: A total of 109 blood samples were collected from patients with acute febrile illness that presented at the Emergency Department of Hospital Universiti Sains Malaysia, Malaysia. Clinical samples were subjected to Leptospira IgM Rapid test, microscopic agglutination test (MAT), isolation by culture method, and direct real-time PCR test. For leptospiral isolation, the samples (whole blood and deposit from spun plasma) were cultured into modified Ellinghausen McCullough Johnson Harris (EMJH) media with and without 5'-fluorouracil (5-FU). In every culture positive sample, partial 16S rRNA gene sequencing was performed for molecular identification of the isolates. Phylogenetic analysis was carried out to determine the genetic relatedness among the isolates. An inhibition of 5-FU study was performed on Leptospirainterrogans serovar Canicola with different concentrations to compare the growth detection of the tested Leptospira with or without 5-FU within 7 days of incubation. Results: Leptospirosis was diagnosed in 14.7% of patients with acute febrile illness. Two Leptospira spp. (n = 2/109, 1.85%) were successfully isolated from whole blood and deposit from spun plasma samples. B004 and B208 samples were positive at day 11 and day 7, respectively, in EMJH media without addition of 5-FU. Sample B004 was identified as Leptospira interrogans and B208 as Leptospira weilli. Phylogenetic analysis confirmed that both of them were within pathogenic group and they were not related. The 5-FU inhibition study revealed that additional of 5-FU at final concentration of 200 µg/mL to EMJH media demonstrated an inhibitory effect on the growth of the tested strain Conclusion: Isolation of Leptospira spp. using EMJH media without addition of 5'-fluorouracil resulted in a better outcome. Two pathogenic Leptospira isolates were successfully cultivated from patients with acute febrile illness that were genetically not related.

Complete Genome Sequence of Leptospira kmetyi LS 001/16, Isolated from a Soil Sample Associated with a Leptospirosis Patient in Kelantan, Malaysia.

The Gram-negative pathogenic spirochetal bacteria Leptospira spp. cause leptospirosis in humans and livestock animals. Leptospira kmetyi strain LS 001/16 was isolated from a soil sample associated with a leptospirosis patient in Kelantan, which is among the states in Malaysia with a high reported number of disease cases. Here, we report the complete genome sequence of Leptospira kmetyi strain LS 001/16.

Duplex TaqMan Hydrolysis Probe-Based Molecular Assay for Simultaneous Detection and Differentiation of Burkholderia pseudomallei and Leptospira spp. DNA.

Melioidosis and leptospirosis, caused by two different bacteria, Burkholderia pseudomallei and Leptospira spp., are potentially fatal infections that share a very similar spectrum of clinical features and cause significant mortality and morbidity in humans and livestock. Early detection is important for better clinical consequences. To our knowledge, there is no diagnostic tool available to simultaneously detect and differentiate melioidosis and leptospirosis in humans and animals. In this study, we described a duplex TaqMan probe-based qPCR for the detection of B. pseudomallei and Leptospira spp. DNA. The performance of the assay was evaluated on 20 B. pseudomallei isolates, 23 Leptospira strains, and 39 other microorganisms, as well as two sets of serially diluted reference strains. The duplex qPCR assay was able to detect 0.02 pg (~ 4 copies) Leptospira spp. DNA and 0.2 pg (~ 25.6 copies) B. pseudomallei DNA. No undesired amplification was observed in other microorganisms. In conclusion, the duplex qPCR assay was sensitive and specific for the detection of B. pseudomallei & Leptospira spp. DNA and is suitable for further analytical and clinical evaluation.

Revisiting the taxonomy and evolution of pathogenicity of the genus Leptospira through the prism of genomics.

The causative agents of leptospirosis are responsible for an emerging zoonotic disease worldwide. One of the major routes of transmission for leptospirosis is the natural environment contaminated with the urine of a wide range of reservoir animals. Soils and surface waters also host a high diversity of non-pathogenic Leptospira and species for which the virulence status is not clearly established. The genus Leptospira is currently divided into 35 species classified into three phylogenetic clusters, which supposedly correlate with the virulence of the bacteria. In this study, a total of 90 Leptospira strains isolated from different environments worldwide including Japan, Malaysia, New Caledonia, Algeria, mainland France, and the island of Mayotte in the Indian Ocean were sequenced. A comparison of average nucleotide identity (ANI) values of genomes of the 90 isolates and representative genomes of known species revealed 30 new Leptospira species. These data also supported the existence of two clades and 4 subclades. To avoid classification that strongly implies assumption on the virulence status of the lineages, we called them P1, P2, S1, S2. One of these subclades has not yet been described and is composed of Leptospira idonii and 4 novel species that are phylogenetically related to the saprophytes. We then investigated genome diversity and evolutionary relationships among members of the genus Leptospira by studying the pangenome and core gene sets. Our data enable the identification of genome features, genes and domains that are important for each subclade, thereby laying the foundation for refining the classification of this complex bacterial genus. We also shed light on atypical genomic features of a group of species that includes the species often associated with human infection, suggesting a specific and ongoing evolution of this group of species that will require more attention. In conclusion, we have uncovered a massive species diversity and revealed a novel subclade in environmental samples collected worldwide and we have redefined the classification of species in the genus. The implication of several new potentially infectious Leptospira species for human and animal health remains to be determined but our data also provide new insights into the emergence of virulence in the pathogenic species.

Reply to Comments on the Study "The Sensitivity, Specificity and Accuracy of Warning Signs in Predicting Severe Dengue, the Severe Dengue Prevalence and its Associated Factors".

Thank you for the comments received on the article "The Sensitivity, Specificity and Accuracy of Warning Signs in Predicting Severe Dengue, the Severe Dengue Prevalence and its Associated Factors" [...].

Probe-specific loop-mediated isothermal amplification magnetogenosensor assay for rapid and specific detection of pathogenic Leptospira.

This study highlighted the performance of the developed integrated loop-mediated isothermal amplification (LAMP) coupled with a colorimetric DNA-based magnetogenosensor. The biosensor operates through a DNA hybridization system in which a specific designed probe captures the target LAMP amplicons. We demonstrated the magnetogenosensor assay by detecting pathogenic Leptospira, which causes leptospirosis. The color change of the assay from brown to blue indicated a positive result, whereas a negative result was indicated by the assay maintaining its brown color. The DNA biosensor was able to detect DNA at a concentration as low as 200 fg/µl, which is equivalent to 80 genomes/reaction. The specificity of the biosensor assay was 100% when it was evaluated with 172 bacterial strains. An integrated LAMP and probe-specific magnetogenosensor was successfully developed, promising simple and rapid visual detection in clinical diagnostics and service as a point-of-care device.

Usefulness of paired samples for the Serodiagnosis of toxoplasmosis infection in a tertiary teaching Hospital in Malaysia.

BACKGROUND: Accurate diagnosis of Toxoplasma gondii (T. gondii) infection remains elusive and requires a comprehensive assessment through laboratory and clinical investigation. In this study, a diagnostic algorithm based on paired serum samples and clinical data was developed and evaluated. METHODS: A total of 1267 suspected cases of Toxoplasma infection were enrolled in this study from January 2016 to December 2016. The cases were screened for anti-Toxoplasma IgM and IgG by electrochemiluminiscence immunoassay (ECLIA) method. Based on the serological profiles, all cases with first seropositive serum samples were considered as suggestive cases of Toxoplasma infection. Thus, second serum samples were obtained after an interval of 2 weeks. The diagnosis was made based on laboratory results and clinical data. RESULTS: A total of 482 T. gondii seroreactive cases were selected. The patient's records were traced and the data were analysed. Accordingly, 152 cases were diagnosed as clinically confirmed cases; 198 cases were clinically asymptomatic and 132 cases were newborn babies or infants who did not have toxoplasmosis and only acquired passive immunity from their mothers. The paired serum algorithm allowed classifying the seroreactive cases as follows: early (0.6%), acute (1.9%), reactivation (13.5%), recent (1.5%), passive immunity from mother (27.3%) and possible congenital infections (1.2%). In addition, cases of reactivated toxoplasmosis were detected among the pregnant mothers (13/82; 15.8%), children aged above 1 year (2/8; 25.0%) and immunocompetent mothers (5/135; 3.7%). Furthermore, the application of the paired serum analysis resulted in remarkably improved treatment initiation. CONCLUSIONS: Toxoplasmosis diagnosis and treatment can be improved through the use of paired serum diagnostic algorithm.

The Sensitivity, Specificity and Accuracy of Warning Signs in Predicting Severe Dengue, the Severe Dengue Prevalence and Its Associated Factors.

Objectives: To study Malaysian dengue clinical practice guideline (CPG) warning signs (WS) in predicting severe dengue (SD) and its associated factors among confirmed cases presented to a teaching hospital in north-eastern Malaysia in 2014. Methods: A cross-sectional study was performed in February 2015 using secondary data acquired from the hospital records. There were 2607 confirmed dengue cases presented to Hospital Universiti Sains Malaysia (HUSM) in 2014. Seven hundred patients were selected after proportionate stratified random sampling conducted according to the number of cases in 12 different months in 2014. Data were collected and analysed using SPSS version 22.0. Results: Severe dengue outcomes represented 4.9% of cases. The prevalence of any of WS in SD was 91.2%. The most common WSs prior to SD were persistent vomiting (55.9%), and abdominal pain/tenderness (52.9%). The most sensitive warning sign in detecting SD was abdominal pain (59%). Specificity of individual WS were generally good, especially of clinical fluid accumulation (99%), hepatomegaly (98%) and mucosal bleeding (93%). Factors associated with SD were persistent vomiting (Adjusted odds ratio (aOR)): 2.41), mucosal bleeding (aOR: 4.73) and haematocrit rise with rapid platelet drop (aOR: 2.74). Conclusion: A focus on sensitivity, specificity, predictive values and association of a number of particular WS should be emphasized in order to better predict severe dengue outcomes.

Leptospirosis seropositivity and its serovars among cattle in Northeastern Malaysia.

BACKGROUND: Leptospirosis is a zoonotic disease that infects human and livestock which causes economic losses to the farmers. It has been reported as one of the causes of reproductive failure in cattle and other ruminants, determining abortions, stillbirth, weak newborns, and decrease in their growth rate and milk production. AIM: The objectives of this study were to determine the leptospirosis seroprevalence and to identify the predominant infecting serovars among cattle. MATERIALS AND METHODS: A cross-sectional study involving 420 cattle from six randomly selected districts in Kelantan was conducted. A serological test using the microscopic agglutination test was conducted in the Institute of Medical Research with a cutoff titer for seropositivity of ≥1:100. RESULTS: The overall prevalence of leptospirosis seropositivity among cattle in this study was 81.7% (95% confidence interval: 63.5, 80.1). The most common reaction obtained with the sera tested was from the serovar Sarawak with 78.8%. CONCLUSION: A high seroprevalence of leptospiral antibodies was found among cattle in Northeastern Malaysia. These findings urge that more studies are required to determine the reasons for the high seroprevalence among the cattle along with its transmission and pathogenicity of the local serovar Sarawak.

Leptospirosis and Workplace Environmental Risk Factors among Cattle Farmers in Northeastern Malaysia.

BACKGROUND: Leptospirosis is an emerging zoonosis and its occurrence has been reported to be rising globally. The environment plays an important role in the survival of Leptospira and determines the risk of infection. Those who were exposed to and had contact with contaminated environment through their occupational, recreational and other activities can be infected with the organism. OBJECTIVE: To determine the seroprevalence of leptospirosis among cattle farmers, prevalence of pathogenic Leptospira, and the workplace environmental risk factors for leptospirosis among cattle farmers in northeastern Malaysia. METHODS: A cross-sectional study involving 120 cattle farmers was conducted. The participants answered an interviewer-guided questionnaire that consisted of sociodemographic and workplace environment characteristics questionnaire, before having their blood sample taken for microscopic agglutination test (MAT). Seropositivity was determined using a cut-off titer of ≥1:100. 248 environmental samples were also collected from the cattle farms for polymerase chain reaction (PCR). RESULTS: The overall seroprevalence of leptospiral antibodies was 72.5% (95% CI 63.5% to 80.1%) and the prevalence of pathogenic Leptospira in the cattle farms environment was 12.1% (95% CI 8.4% to 17.0%). The independent factors associated with seropositivity of leptospirosis among cattle farmers were positive pathogenic Leptospira in the environment (Adj OR 5.90, 95% CI 1.34 to 26.01) and presence of garbage dumping in the farm (Adj OR 2.40, 95% CI 1.02 to 5.65). CONCLUSION: Preventing leptospirosis incidence among cattle farmers necessitates changes in work environment. Identifying modifiable factors may also contribute to the reduction of infection.

Development and validation of pan-Leptospira Taqman qPCR for the detection of Leptospira spp. in clinical specimens.

BACKGROUND: Early diagnosis of leptospirosis is important for ensuring better clinical management and achieving better outcomes. Currently, serological assays suffer from inconsistent performance and are less useful for early diagnosis of leptospirosis. As an alternative, qPCR is more sensitive, specific and able to detect the presence of leptospiral DNA during the acute phase of the infection. Meanwhile, most molecular assays do not detect the non-pathogenic group of Leptospira, even though these groups may also infect humans, although less frequently and less severely. METHODS: A set of primers and probe targeting rrs genes of 22 Leptospira spp. were designed and evaluated on 31 Leptospira isolates, 41 other organisms and 65 clinical samples from suspected patients. RESULTS: The developed assay was able to detect as low as 20 fg Leptospira DNA per reaction (equivalent to approximately 4 copies) and showed high specificity against the tested leptospiral strains. No cross amplification was observed with the other organisms. During the evaluation of the confirmed clinical specimens, the developed assay was able to correctly identify all positive samples (n = 10/10). One amplification was observed in a negative sample (n = 1/55). The sequencing of the PCR product of the discordant sample revealed that the sequences were similar to those of L. interrogans and L. kirschneri. CONCLUSION: The findings suggest that the developed Taqman qPCR assay is sensitive, specific and has potential to be applied in a larger subsequent study.

Anti-microbial Activity of Aqueous Quercus infectoria Gall Extract against Pathogenic Leptospira.

BACKGROUND: Quercus infectoria gall extract is known to have broad spectrum anti-microbial activity in vitro. This study was conducted to determine the anti-microbial activity of Q. infectoria gall extract against pathogenic Leptospira and to evaluate the morphological changes of extract-treated cells using a scanning electron microscope (SEM). METHODS: A two-fold serial microdilution broth assay was used to determine the minimum inhibitory concentration (MIC) of aqueous Q. infectoria gall extract against the L. interrogans serovar Javanica and the L. interrogans serovar Icterohaemorrhagiae, at concentrations ranging from 4.00 mg/mL to 0.0078 mg/mL. The minimum bactericidal concentration (MBC) was determined by sub-culturing the broth from the microtiter plate wells that showed no apparent growth or turbidity to the freshly prepared Ellinghausen-McCullough-Johnson-Harris (EMJH) broth media, and it was subsequently observed under a dark field microscope following three weeks of incubation for purposes of growth detection. The cell morphology of both extract-treated and untreated L. interrogans serovar Icterohaemorhagiae was analysed using the SEM. RESULTS: The results of the broth microdilution assay demonstrate that the aqueous Q. infectoria gall extract possesses anti-microbial activity against both of the L. interrogans serovars with MIC values of 0.125 mg/mL. The MBC values for the L. interrogans serovar Javanica and the L. interrogans serovar Icterohaemorhagiae are 0.125 mg/mL and 0.250 mg/mL, respectively. The SEM micrograph shows changes in shape and size of the extract-treated cells (at 8× MIC) in comparison to the untreated cells. CONCLUSION: The Q. infectoria gall extract displays anti-microbial inhibition and killing activity against the pathogenic Leptospira isolates, and thus has the potential for further exploration of its efficacy and use in the treatment of leptospirosis.