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Could rapid test reader maximize the benefits to the HIV self-tester?

Balakrishnan P, Girija ASS, Kannan I, Vignesh R, Shankar EM, Sucharitha ST

Indian J Med Microbiol, 2023;43:49-50.
PMID: 36280566 DOI: 10.1016/j.ijmmb.2022.10.004
Fulltext ermA, ermC , tetM and tetK are essential for erythromycin and tetracycline resistance among methicillin-resistant Staphylococcus aureus strains isolated from a tertiary hospital in Malaysia

Lim KT, Hanifah YA, Yusof M, Thong KL

Indian J Med Microbiol, 2012 Apr-Jun;30(2):203-7.
PMID: 22664438 DOI: 10.4103/0255-0857.96693

The objective of this study was to determine the expression and transferability of tetracycline and erythromycin resistance among 188 MRSA strains from a Malaysian tertiary hospital. The minimum inhibitory concentrations (MICs) for oxacillin, erythromycin, tetracycline and ciprofloxacin ranged from 4 to 512 μg/ml, 0.25 to 256 μg/ml, 0.5 to 256 μg/ml and 0.5 to 512 μg/ml, respectively. Tetracycline-resistant strains showed co-resistance towards ciprofloxacin and erythromycin. There was a significant increase (P<0.05) of high-level tetracycline (≥MIC 256 μg/ml) and erythromycin (≥MIC 128 μg/ml) resistant strains in between the years 2003 and 2008. All erythromycin-resistant strains harboured ermA or ermC gene and all tetracycline-resistant strains harboured tetM or tetK gene. The blaZ was detected in all MRSA strains, whereas ermA, tetM, ermC, tetK and msrA genes were detected in 157 (84%), 92 (49%), 40 (21%), 39 (21%) and 4 (2%) MRSA strains, respectively. The blaZ, tetM, ermC and tetK genes were plasmid-encoded, with ermC gene being easily transmissible. Tn5801-like transposon was present in 78 tetM-positive strains. ermA and tetM genes were the most prevalent erythromycin and tetracycline resistance determinants, respectively, in MRSA strains. The association of resistance genes with mobile genetic elements possibly enhances the spread of resistant traits in MRSA.
Fulltext Stacking gels: A method for maximising output for pulsed-field gel electrophoresis

Heng SK, Heng CK, Puthucheary SD

Indian J Med Microbiol, 2009 Apr-Jun;27(2):142-5.
PMID: 19384038 DOI: 10.4103/0255-0857.49428

Pulsed field gel electrophoresis (PFGE), the gold standard of molecular typing methods, has a major disadvantage of an unusually long electrophoretic time. From the original protocol of 6 days, it was modified to 3 days and subsequently to a single day. We describe the procedure of stacking five to six gels one on top of another in order to increase and maximize the output in a shorter time without compromising the resolution and reproducibility. All the variables that affect pulsed field gels during electrophoresis were taken into consideration. We firstly optimized the parameters to be used and secondly determined whether stacking of five to six gels had any effect on the molecular separation during electrophoresis in comparison with a single gel run. DNA preparation, restriction, electrophoresis, staining and gel documentation was carried out based on previously published methods. Gels were analysed using BioNumerics and dice coefficient and unweighted pair group methods were used to generate dendrograms based on 1.5% tolerance values. Identical band profiles and band resolution-separation were seen in the PFGE patterns with single gel and multiple stacking gels. Cluster analysis further strengthened the fact that results from stacking gels were reproducible and comparable with a single gel run. This method of stacking gels saves time and maximizes the output at the same time. The run time for a single gel was about 28 hours, but with six stacked gels the run time was 54 hours compared with 28 x 6 = 168 hours if they were run separately as single gels thus saving time of 67.86%. Beside the big factor of saving time, stacking gels save resources (electricity, reagents, water, chemicals and working time) by increasing the sample throughput in a shorter time without compromising on quality of data. But optimization of working parameters is vital depending on the PFGE system used.
Molecular characterization of genes encoding the quinolone resistance determining regions of Malaysian Streptococcus pneumoniae strains

Kumari N, Subramaniam G, Navaratnam P, Sekaran SD

Indian J Med Microbiol, 2008 5 1;26(2):148-50.
PMID: 18445951

Genes encoding the quinolones resistance determining regions (QRDRs) in Streptococcus pneumoniae were detected by PCR and the sequence analysis was carried out to identify point mutations within these regions. The study was carried out to observe mutation patterns among S. pneumoniae strains in Malaysia. Antimicrobial susceptibility testing of 100 isolates was determined against various antibiotics, out of which 56 strains were categorised to have reduced susceptibility to ciprofloxacin (>or=2 microg/mL). These strains were subjected to PCR amplification for presence of the gyrA, parC , gyrB and parE genes. Eight representative strains with various susceptibilities to fluoroquinolones were sequenced. Two out of the eight isolates that were sequenced were shown to have a point mutation in the gyrA gene at position Ser81. The detection of mutation at codon Ser81 of the gyrA gene suggested the potential of developing fluoroquinolone resistance among S. pneumoniae isolates in Malaysia. However, further experimental work is required to confirm the involvement of this mutation in the development of fluoroquinolone resistance in Malaysia.
Rapid detection of non-enterobacteriaceae directly from positive blood culture using fluorescent in situ hybridization

Wong EH, Subramaniam G, Navaratnam P, Sekaran SD

Indian J Med Microbiol, 2007 Oct;25(4):391-4.
PMID: 18087092

Fluorescent in situ hybridization (FISH) was carried out using two different oligonucleotide probes specific for Pseudomonas spp. and Acinetobacter spp. These probes were tested against different organisms and were found to be highly specific. Sensitivity testing showed that the probes were able to detect as low as 10 3 CFU/mL. In addition, FISH was carried out directly on positive blood culture samples and the detection of microorganisms took less than 2 h. We believe that FISH is a rapid method that can be used as a routine laboratory diagnostic technique for the detection of Acinetobacter spp. and Pseudomonas spp. in clinical samples.
SHV-5 extended-spectrum beta-lactamases in clinical isolates of Escherichia coli in Malaysia

Subramaniam G, Palasubramaniam S, Navaratnam P

Indian J Med Microbiol, 2006 Jul;24(3):205-7.
PMID: 16912441

Escherichia coli isolates resistant to ceftazidime isolated in the University Malaya Medical Center (UMMC) Kuala Lumpur, Malaysia, between the years 1998 and 2000 were studied for extended-spectrum beta-lactamase (ESBL) production. All strains were analysed phenotypically and genotypically and found to be ESBL-producing organisms harbouring SHV-5 beta-lactamase. This was confirmed by PCR-SSCP and nucleotide sequencing of the blaSHV amplified gene. As there was no evidence of ESBL activity in E. coli prior to this, coupled with the fact that there was a predominance of SHV-5 beta-lactamases in Klebsiella pneumoniae isolates in UMMC, we postulate that the E. coli obtained the SHV-5 beta-lactamase genes by plasmid transfer from the ESBL-producing K. pneumoniae.
Survey of Staphylococcus isolates among hospital personnel, environment and their antibiogram with special emphasis on methicillin resistance

Shobha KL, Rao PS, Thomas J

Indian J Med Microbiol, 2005 Jul;23(3):186-8.
PMID: 16100427

The objective of this study was to find the prevalence of Staphylococcus spp. carriage among hospital personnel and hospital environment and their antibiogram with special emphasis on methicillin resistance. A total of 205 samples from hospital personnel and environment were collected from casualty, oncology and multidisciplinary cardiac unit ward of Kasturba Medical College Hospital, Manipal. Samples were collected using sterile cotton wool swabs and inoculated into brain heart infusion broth. Subcultures were done onto blood agar and MacConkey's agar. Isolates were identified by standard methods up to species level. Antimicrobial susceptibility test was performed according to standardized disc diffusion Kirby-Bauer method. Each of the isolates was screened for methicillin resistance using oxacillin disc on Mueller Hinton agar plate followed by MIC for methicillin and cefoxitin susceptibility test by disc diffusion method. Sixty five out of 205 strains (31.7%) were Staphylococcus spp. and all of them were coagulase negative. Most of the strains belonged to S.epidermidis 49.23% (32/65) followed by S. saprophyticus 26.15% (17/65). Maximum isolates of S.epidermidis were from anterior nares 28.12% (9/32 strains of S.epidermidis). Highest number of methicillin resistant coagulase negative strains (3/9, 33.33%) were isolated from stethoscope of multidisciplinary cardiac unit ward followed by carriers in the anterior nares (2/9, 22.22%). Methicillin resistant coagulase negative staphylococci are prevalent in anterior nares of hospital personnel and in the hospital environment thereby providing a definite source for hospital acquired infection. All isolates were sensitive to vancomycin, ciprofloxacin and amikacin.
Fulltext Antimicrobial susceptibility of Leptospira spp. isolated from environmental, human and animal sources in Malaysia

Benacer D, Zain SN, Ooi PT, Thong KL

Indian J Med Microbiol, 2017 Jan-Mar;35(1):124-128.
PMID: 28303833 DOI: 10.4103/ijmm.IJMM_15_458

Leptospirosis is a zoonosis with worldwide distribution caused by pathogenic spirochetes of the genus Leptospira. The aim of this study was to evaluate the susceptibility of isolates obtained from different hosts. A total of 65 Leptospira isolates from humans (n = 1), zoonoses (rat, n = 60; dog, n = 1; swine, n = 1) and environment (n = 2) were tested against six antibiotics. All the isolates were resistant to trimethoprim and sulphamethoxazole and had high MIC toward chloramphenicol (MIC90: 6.25 μg/ml). All except one environment isolate were sensitive to ampicillin, doxycycline and penicillin G.
Fulltext Occurrence of enteric parasitic infections among HIV-infected individuals and its relation to CD4 T-cell counts with a special emphasis on coccidian parasites at a tertiary care centre in South India

Swathirajan CR, Vignesh R, Pradeep A, Solomon SS, Solomon S, Balakrishnan P

Indian J Med Microbiol, 2017 Jan-Mar;35(1):37-40.
PMID: 28303816 DOI: 10.4103/ijmm.IJMM_16_164

CONTEXT: Diarrhoea is one of the major complications occurring in over 90% of HIV-infected individuals in developing countries. Coccidian group of parasites, being opportunistic pathogens, have been implicated as the most common causative agents of diarrhoea among HIV-infected population.

AIMS: The aim was to study the magnitude of parasitic diarrhoea with special context to coccidian parasitic infections in HIV-infected individuals and their association with the patient's immunological status measured by CD4 T-cell counts.

SETTINGS AND DESIGN: This investigation was performed between January 2002 and December 2014 at a tertiary HIV care centre in Chennai, South India.

MATERIALS AND METHODS: Stool samples were collected and microscopically observed for parasites using direct, formal-ether-concentrated wet mounts and modified acid-fast staining for coccidian parasites. CD4 T-cell counts were done by FACScount.

STATISTICAL ANALYSIS USED: All statistical analyses were performed using GraphPad Prism software, version 5.0, andP < 0.05 was considered statistically significant.

RESULTS: Coccidian parasitic infection accounted for about 23.4% of parasitic infections, and of these, Cystoisospora belli was observed to be the most common cause of diarrhoea (88.8%), followed by Cryptosporidium spp. (9.9%) and Cyclospora spp. (1.3%). Trend analysis of coccidian aetiology during the study period revealed a significant rise in the positivity of C. belli and Cryptosporidium spp. (P = 0.001). Among the HIV patients with CD4+ T-cell counts <200 cells/μL, Cryptosporidium infection was most common (90%), followed by infection with C. belli(61.4%).

CONCLUSIONS: Coccidian parasites continue to be the most common aetiological agent of diarrhoea among patients with HIV. The increasing trend of positivity of both cystoisosporiasis and cryptosporidiosis over the study period and the high positivity of cryptosporidiosis in patients with lower CD4+ T-cell counts are issues of serious concern. The findings call for the need for the early diagnosis of coccidian parasites and appropriate intervention among HIV-infected patients.
Fulltext Risk factors and frequency of tuberculosis-associated immune reconstitution inflammatory syndrome among HIV/Tuberculosis co-infected patients in Southern India

Vignesh R, Swathirajan CR, Solomon SS, Shankar EM, Murugavel KG

Indian J Med Microbiol, 2017 Apr-Jun;35(2):279-281.
PMID: 28681821 DOI: 10.4103/ijmm.IJMM_16_163

Immune reconstitution inflammatory syndrome (IRIS) continues to be a complication in HIV/tuberculosis (TB) co-infected patients initiating highly active antiretroviral therapy (HAART). The aim of this study was to evaluate the risk factors associated with developing IRIS to identify a possible biomarker to predict or diagnose IRIS in patients initiating HAART. A total of 175 HIV/TB co-infected patients initiating HAART were followed up longitudinally during September 2010 to May 2013 attending a HIV care clinic in Chennai. Patients were followed up longitudinally after HAART initiation and baseline demographic, laboratory parameters and treatment characteristics between patients with IRIS events and those without IRIS events were compared. Chi-square or Fisher's exact test for categorical variables and a Wilcoxon rank-sum test for continuous variables were performed using SPSS, version 12.0 software. Patients with IRIS had a significantly lower median baseline CD4+ T-cell count (P = 0.0039). There were no differences in terms of sex, CD4 T-cell %, plasma viral load, time interval between initiating ATT and HAART between the IRIS and non-IRIS patients. Low CD4+ T-cell count (<100 cells/μL) could be used as a marker to screen and monitor patients initiating HAART.
Fulltext Identification of multiple strains of Porphyromonas gingivalis using heteroduplex polymerase chain reaction in varying severity of chronic periodontitis

Kulkarni MR, Bhat KG, Thomas BS, Bhat GS, Kulkarni RD

Indian J Med Microbiol, 2018 5 8;36(1):81-86.
PMID: 29735832 DOI: 10.4103/ijmm.IJMM_17_434

Aim: Research has demonstrated that there are multiple strains of Porphyromonas gingivalis with varying potency to cause periodontal disease. The current study aims at using heteroduplex polymerase chain reaction (PCR) to detect the strain diversity of P. gingivalis in periodontitis lesions of varying severity in a sample of the Indian population.
Materials and Methods: Subgingival plaque samples were collected from 60 individuals with varying severity of chronic periodontitis and 30 individuals with a clinically healthy periodontium. The samples were subjected to PCR analysis to identify P. gingivalis, followed by heteroduplex analysis to identify the strain diversity in a given sample. Bacterial culture was carried out as a comparative standard.
Results: Of the 56 samples that were positive for P. gingivalis by PCR, 54 samples yielded eight different heteroduplex patterns. Analysis of these patterns indicated that two strains of P. gingivalis were present in 41 individuals (45.6%) and three strains were present in 13 individuals (14.4%). Detection of P. gingivalis by PCR was significantly more in the periodontitis group as compared to the healthy group.
Conclusions: Species-specific PCR and heteroduplex analysis provide a simple and accurate method to analyse the strain diversity of P. gingivalis. P. gingivalis was detected in both healthy periodontal sites as well as sites with periodontitis. The presence of two or three P. gingivalis strains was seen in 60% of the samples.
Comparative study of antifungal susceptibility testing methods for clinical Candida albicans isolates

Tan XT, Mohd Shuhairi NB, Mohd Tap R, Amran F

Indian J Med Microbiol, 2024 Jul 05;50:100663.
PMID: 38964501 DOI: 10.1016/j.ijmmb.2024.100663

PURPOSE: Candida albicans is the second most common cause of candidemia in Malaysia. The Clinical and Laboratory Standards Institute (CLSI) broth microdilution method is the gold standard for determining its minimum inhibitory concentration (MIC); however, it is laborious and time-consuming. This study was conducted to evaluate the usefulness of alternative methods, namely Sensititre YeastOne (SYO), VITEK 2 system, and E-test for determining the MIC of clinical C. albicans isolates.
MATERIALS AND METHODS: The susceptibilities of 95 C. albicans isolates were compared between SYO, VITEK 2 system, and E-test with CLSI broth microdilution method. The categorical agreement (CA), essential agreement (EA), very major errors (VME), major errors (ME) and minor errors (MiE) were calculated.
RESULTS: Our finding showed the CA varied for SYO from 96.8% to 100%, while the EA ranged from 91.6% to 100%. The SYO method showed 1.1% of VME and ME, and up to 3.2% of MiE. Next, the CA and EA ranges for the VITEK 2 system were 97.8%-100% and 23.2%-100%, respectively. In the VITEK 2 technique, 1.1% of VME were found. For the E-test, the CA varied from 83.2% to 100% while the EA ranged from 64.2% to 98.9%. The E-test method showed 1.1% of VME and up to 16.8% of MiE.
CONCLUSIONS: In conclusion, SYO and VITEK 2 (except flucytosine) could be potential alternatives to the CLSI broth microdilution method in determining the MIC of C. albicans.

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