Andi R. Sultan

In vitro induction of NETosis: Comprehensive live imaging comparison and systematic review

Background Multiple inducers of in vitro Neutrophil Extracellular Trap (NET) formation (NETosis) have been described. Since there is much variation in study design and results, our aim was to create a systematic review of NETosis inducers and perform a standardized in vitro study of NETosis inducers important in (cardiac) wound healing. Methods In vitro NETosis was studied by incubating neutrophils with PMA, living and dead bacteria (S. aureus and E. coli), LPS, (activated) platelets (supernatant), glucose and calcium ionophore Ionomycin using 3-hour periods of time-lapse confocal imaging. Results PMA is a consistent and potent inducer of NETosis. Ionomycin also consistently resulted in extrusion of DNA, albeit with a process that differs from the NETosis process induced by PMA. In our standardized experiments, living bacteria were also potent inducers of NETosis, but dead bacteria, LPS, (activated) platelets (supernatant) and glucose did not induce NETosis. Conclusion Our systematic review confirms that there is much variation in study design and results of NETosis induction. Our experimental results confirm that under standardized conditions, PMA, living bacteria and Ionomycin all strongly induce NETosis, but real-time confocal imaging reveal different courses of events.

Detection and identification of mycobacteria in sputum from suspected tuberculosis patients

BackgroundDetection of Tuberculosis agent like nontuberculous mycobacteria (NTM) species by culture and microscopic methods remains difficult and time consuming. A fast and reliable diagnosis of tuberculosis would greatly improve the control of the disease. The purpose of this study is to compare the conventional multiplex PCR and multiplex PCR reverse cross blot hybridization assay to culture method in terms of mycobacteria species detection.FindingsAmong the 117 positively cultured samples, nontuberculous mycobacteria (NTM) species were found in 9 samples of multiplex PCR reverse cross blot hybridization assay; compared to only 3 NTM species found in our conventional multiplex PCR, and 13 NTM species were successfully identified among 162 negatively cultured samples compared to only 5 NTM species identification in conventional multiplex PCR results.ConclusionsThe sensitivity of the multiplex PCR reverse cross blot hybridization assay comparing to culture method was 86.03%, the specificity is 35.46%, the positive predictive value was 41.94% and the negative predictive value was 82.41%. For conventional multiplex PCR these values are 81.62%, 38.65%, 41.89%, 79.51%, respectively. Furthermore, in terms of mycobacteria species detection, the conventional multiplex PCR was relatively equal compared to the multiplex PCR reverse cross blot hybridization assay, and to be particularly having no significant discrepant results on the identification of Mycobacteria tuberculosis in both methods.

Multi-Locus Variable-Number Tandem Repeat Profiling of Salmonella enterica Serovar Typhi Isolates from Blood Cultures and Gallbladder Specimens from Makassar, South-Sulawesi, Indonesia

Multi-locus variable-number tandem repeat analysis differentiated 297 Salmonella enterica serovar Typhi blood culture isolates from Makassar in 76 genotypes and a single unique S. Typhi genotype was isolated from the cholecystectomy specimens of four patients with cholelithiasis. The high diversity in S. Typhi genotypes circulating in Makassar indicates that the number of carriers could be very large, which may complicate disease prevention and control.

Sccmec type II gene is common among clinical isolates of methicillin-resistant Staphylococcus aureus in Jakarta, Indonesia.

BackgroundCommunity Acquired Methicillin Resistant Staphylococcus aureus (CA-MRSA) is a strain of MRSA that can cause infections in patients in the community, in which these patients had no previous risk factors for MRSA infection and the patient received 72 hours prior to infection when admitted to hospital. This study aims to determine and compare the characteristics of epidemiological, clinical, and molecular biology of CA-MRSA with HA-MRSA.MethodsA total of 11 clinical strains of Methicillin-resistant Staphylococcus aureus (MRSA) and Methicillin-sensitive Stapylococcus aureus (MSSA) were collected from 2 hospitals in Jakarta, Indonesia in 2012. SCCmec typing was performed by multiplex polymerase chain reaction (PCR) and the presence of six genes (vraR, vraG, vraA, vraF,fruA, and fruB) associated with vancomycin resistance was examined by simple PCR analysis.ResultsWe found three strains of community-acquired MRSA with SCCmec type II and one strain of hospital-acquired MRSA with SCCmec type IV. The other seven strains did not contain mecA genes and SCCmec. Plasmid pUB110 was found in one strain of community-acquired MRSA and two strains of hospital-acquired MRSA. vraA genes were present in 9 of the 11 strains, vraF in 4, vraG in 5, and vraR in 4. Note worthily, three quarters of strains without pUB110 contained vraR and vraF, and 70% contained vraA, whereas 60% of strains with pUB110 contained vraG.ConclusionBased on these results, we should be concerned about the possibility of transition from MRSA strains sensitive to vancomycin in VISA strains of MRSA strains obtained in clinical trials. But first we need to look the existence of natural VISA or hVISA among these MRSA strains.

Risk Factors of Typhoid Infection in the Indonesian Archipelago

Background Knowledge of risk factors and their relative importance in different settings is essential to develop effective health education material for the prevention of typhoid. In this study, we examine the effect of household level and individual behavioural risk factors on the risk of typhoid in three Indonesian islands (Sulawesi, Kalimantan and Papua) in the Eastern Indonesian archipelago encompassing rural, peri-urban and urban areas. Methods We enrolled 933 patients above 10 years of age in a health facility-based case-control study between June 2010 and June 2011. Individuals suspected of typhoid were tested using the typhoid IgM lateral flow assay for the serodiagnosis of typhoid fever followed by blood culture testing. Cases and controls were defined post-recruitment: cases were individuals with a culture or serology positive result (n = 449); controls were individuals negative to both serology and culture, with or without a diagnosis other than typhoid (n = 484). Logistic regression was used to examine the effect of household level and individual level behavioural risk factors and we calculated the population attributable fraction (PAF) of removing each risk significant independent behavioural risk factor. Results Washing hands at critical moments of the day and washing hands with soap were strong independent protective factors for typhoid (OR = 0.38 95% CI 0.25 to 0.58 for each unit increase in hand washing frequency score with values between 0 = Never and 3 = Always; OR = 3.16 95% CI = 2.09 to 4.79 comparing washing hands with soap sometimes/never vs. often). These effects were independent of levels of access to water and sanitation. Up to two thirds of cases could be prevented by compliance to these practices (hand washing PAF = 66.8 95% CI 61.4 to 71.5; use of soap PAF = 61.9 95%CI 56.7 to 66.5). Eating food out in food stalls or restaurant was an important risk factor (OR = 6.9 95%CI 4.41 to 10.8 for every unit increase in frequency score). Conclusions Major gains could potentially be achieved in reducing the incidence of typhoid by ensuring adherence to adequate hand-washing practices alone. This confirms that there is a pivotal role for ‘software’ related interventions to encourage behavior change and create demand for goods and services, alongside development of water and sanitation infrastructure.

Diagnostic Evaluation of Tuberculosis

Tuberculosis is still one of the leading causes of death by infectious diseases with 2 million deaths per year and 9.2 million new cases of tuberculosis disease annually [1-3]. Besides, more than 2 milliard people are infected with latent tuberculosis infection (LTBI) [1-3]. Despite continuous effort in the prevention, monitoring and treatment of tuberculosis, the disease remains a major health problem in many countries [4-6], particularly in developing countries like Indonesia [7]. National tuberculosis programs and other programs conducted by foreign organizations still fail to eliminate the transmission and incidence of tuberculosis. Transmis‐ sion is even on the rise in developing countries despite the availability of effective therapies for tuberculosis, whereas the spread and the incidence of tuberculosis in Europe and North America are under control. Several reasons may be responsible for this failure, such us the difficulty of providing adequate anti-tuberculosis medication in many developing countries due to cost issues, the emergence of multi-drug resistant (MDR) strains of M. tuberculosis, and the dramatically high co-incidence of tuberculosis in HIV-infected patients [2, 7]. Another important issue is delay of diagnosis due to the lack of a proper method to identify tuberculosis agents [1, 8].

Staphylococcal protein a is a key factor in neutrophil extracellular traps formation

Staphylococcus aureus are strong inducers of neutrophil extracellular traps (NETs), a defense mechanism of neutrophils against pathogens. Our aim was to explore the role of Protein A in S. aureus-induced NETosis. We determined the Protein A production of four different S. aureus strains and found a direct relationship between the degree of NETosis induction and Protein A production: strains producing higher concentrations of Protein A evoke significantly more NETs. A S. aureus strain in which Protein A as well as a second binding protein for immunoglobulins (Sbi) have been knocked-out (ΔSpA ΔSbi) induced significantly less NETosis than the wild-type strain. NETosis induction by this knockout strain can be rescued by the addition of purified Protein A. Dead S. aureus did not induce NETosis. In conclusion, Protein A is a determinant for NETosis induction by S. aureus.