Molecular detection of drug-resistant Plasmodium falciparum mutants in Assam

Background & objectives: The spread of drug-resistant Plasmodium falciparum ( Pf) poses a serious threat to the control and elimination of malaria. The objective of this study was to detect the molecular biomarkers of antimalarial drug resistance in Pf in patients visiting a tertiary care hospital in Assam. Methods: Malaria was first detected in fever cases using microscopy and a rapid diagnostic test (RDT), and then confirmed using PCR. Pf chloroquine resistance transporter (Pfcrt), Pf multidrug resistance-1 (Pfmdr-1), and single-nucleotide polymorphisms linked to delayed parasite clearance after treatment with artemisinin MAL 10-688956 and MAL 13-1718319 and Kelch-13 propeller (PfK-13) genes were evaluated by PCR-restriction fragment length polymorphism (RFLP). Results: Sixty nine cases of malaria were found among 300 cases of fever. Of these, 54 were positive for Pf, 47 of which were confirmed by PCR. Pfcrt-K76T mutation was seen in 96.6 per cent and Pfmdr1-N86Y mutation in 84.2 per cent of cases. Mutation was not detected in MAL10 and MAL13 genes. Sequence analysis of Kelch-13 gene showed the presence of a novel mutation at amino acid position 675. Statistically, no significant association was found between the molecular biomarkers and demographic profile, clinical presentation and outcome of the cases. Interpretation & conclusions: Molecular surveillance is essential to assess the therapeutic efficacy of the drugs against circulating Pf isolates in Assam which are found to be highly resistant to CQ. The role of the new mutation found in the Kelch-13 gene in the development of artemisinin resistance in Assam needs to be thoroughly monitored in future research.

Species distribution & antifungal susceptibility pattern of oropharyngeal Candida isolates from human immunodeficiency virus infected individuals.

Background & objectives: The changing spectrum of Candida species in causation of oropharyngeal candidiasis and their antifungal susceptibility pattern among the HIV infected individuals has made the identification to species level mandatory and detection of drug resistance necessary for patient care. The present study was carried out to determine the species distribution and antifungal susceptibility profile of oral Candida isolates colonizing or infecting both HIV seropositive and seronegative individuals. Methods: A case-control study was conducted including 141 consecutive, non-repeat HIV-seropositive individuals and an equal number of sex and age matched HIV-seronegative control. Speciation of the oropharyngeal Candida isolates was done using standard yeast identification protocol. Antifungal susceptibility testing was done by the disk-diffusion method as well as by Fungitest method. Results: From the 59 culture positive HIV seropositive cases, 61 Candida isolates were recovered; Candida albicans (n=47, 77.0%), C. dubliniensis (n=9, 14.7%), C. parapsilosis (n=2, 3.2%), C. glabrata (n=2, 3.2%), and C. famata (n=1, 1.6%). Candida colonization in HIV-seropositive individuals was significantly higher than that of HIV-seronegative (control) group. Antifungal susceptibility testing revealed (n=6, 9.3%) C. albicans isolates resistant to voriconazole and fluconazole by disk-diffusion method whereas no resistance was seen by Fungitest method. Interpretation & conclusions: C. albicans was the commonest Candida species infecting or colonizing HIV seropositive individuals. Oropharyngeal Candida isolates had high level susceptibility to all the major antifungals commonly in use. Increased level of immunosuppression in HIV-seropositives and drug resistance of non-albicans Candida species makes identification and susceptibility testing of Candida species necessary in different geographical areas of the country.

Bacterial Pathogens Associated with Community-acquired Pneumonia in Children Aged Below Five Years.

Objectives: To determine the spectrum of bacterial pathogens causing community-acquired pneumonia in children below 5 years of age. Methods: Children aged below 5 years satisfying the WHO criteria for pneumonia, severe pneumonia or very severe pneumonia, and with the presence of lung infiltrates on chest X-ray were enrolled. Two respiratory samples, one for culture and the other for PCR analysis, and a blood sample for culture were collected from every child. Results: Of the 180 samples processed, bacterial pathogens were detected in 64.4%. Streptococcus pneumoniae and Hemophilus influenzae were most frequently detected. The performance of PCR analysis and culture were identical for the typical bacterial pathogens; atypical pathogens were detected by PCR analysis only. Conclusion: S. pneumoniae and H. influenza were the most commonly detected organisms from respiratory secretions of children with community acquired pneumonia.

An appraisal of clinicopathological parameters in Japanese encephalitis and changing epidemiological trends in upper Assam, India.

Context: Japanese encephalitis (JE), an acute mosquito-borne viral disease, is one of the leading causes of viral encephalitis in the South-East Asian region. JE is endemic in Assam. The morbidity and mortality due to JE is significant with outbreaks every year during the monsoons. Aims: The aim was to study the clinicopathological profile of JE; to examine their role in predicting disease outcome; and to document the increase in the incidence of JE among the adult population in this region. Materials and Methods: Clinically suspected acute encephalitis syndrome (AES) cases admitted in Assam Medical College and Hospital during the period of May 2011 to April 2012 were tested by JE virus specific Immunoglobulin M capture ELISA. Statistical Analysis Used: Data analysis was performed using SPSS version 16.0. Results: Out of 424 AES cases, 194 were JE positive. The occurrence of JE in adults was higher (P < 0.001) than the pediatric age group. The study recorded a high rate of renal dysfunction in JE cases. A single case of JE induced abortion and two cases of JE-neurocysticercosis coinfections were documented. Regression analysis revealed that adult population, unconsciousness, paresis and elevated cerebrospinal fluid protein level were associated with a worse prognosis in JE cases. Mortality in JE positive cases was higher than the JE negative cases (P = 0.001). Conclusion: The study attempts to highlight the role played by a combination of clinical and laboratory parameters in assessing the severity and outcome in JE and may help in directing the limited medical resources toward those that need it the most.

Drug resistant Shigella flexneri in & around Dibrugarh, north-east India.

Background & objectives: Shigella flexneri is the most common species of Shigella causing diarrhoea and dysentery in Asia including India. Multidrug resistance in Shigella species has been reported worldwide and there is rising concern regarding development of fluoroquinolone resistance. This study was undertaken to find out the resistance pattern of Sh. flexneri, the commonest shigella isolated in Dibrugarh, north east India, including detection of fluoroquinolone resistance and extended spectrum beta lactamases. Methods: Stool samples collected from patients of diarrhoea and dysentery were tested for bacterial enteropathogens. Strains of Shigella species were confirmed by biochemical tests. Speciation was done using commercially available polyvalent antiserum. Antimicrobial susceptibility test was performed by Kirby Bauer disc diffusion method against 18 different antibiotics. Extended spectrum beta lactamase (ESBL) detection was done by disc approximation test as well as combination disc method and minimum inhibitory concentrations (MIC) of different antibiotics were also measured. Results: Multidrug resistance in Sh. flexneri was found to be common (90.2%) and the commonest phenotypic multi-drug resistance profile was ampicillin-tetracycline-co-trimoxazole-nalidixic acid. High resistance to nalidixic acid was detected in 90.3 per cent isolates (MIC >240 μg/ml) and ciprofloxacin resistance was seen emerging in this region (11.2%, MIC >4 μg/ml). Present of ESBL was phenotypically confirmed in two cases. Besides the fluoroquinolones, chloramphenicol, piperacillin-tazobactum and the third generation cephalosporins were effective in 87-100 per cent of the isolates. Interpretation & conclusions: Our study showed high resistance (MIC >240 μg/ml) against nalidixic acid in Sh. flexneri isolates. Ciprofloxacin resistance is also emerging in this region. Shigellosis due to ESBL carrying Shigella can become a serious threat to public health. Guidelines for therapy should be monitored and modified based on regional reports of resistance to antimicrobial agents.

Mycotic corneal ulcers in upper Assam.

Purpose: To study the association of various risk factors and epidemiological variables of mycotic keratitis treated at a tertiary referral hospital of upper Assam. Materials and Methods: In this hospital-based prospective study a total of 310 consecutive corneal ulcer cases attending the ophthalmology outpatient department of Assam Medical College were enrolled between April 2007 and March 2009. After clinical and slit-lamp biomicroscopic examination in all suspected cases, smears and culture examination for fungus was done to establish the etiology. Demographic information and associated probable risk factors of individual cases were noted in a predesigned questionnaire. Results: In 188 (60.6%) cases fungal etiology could be established. Out of them 67.6% were males. The most commonly affected age group was 41-50 years (25.5%). The maximum (23.4%) cases were reported during the paddy harvesting season in Assam (January and February). Fungal element could be demonstrated in 65.2% cases in direct potassium hydroxide (KOH) mount. The commonest predisposing factor was corneal injury (74.5%). While diabetes was a significant systemic predisposing factor in mixed bacterial and fungal infections in 11.1% cases, blocked naso-lacrimal duct was the local predisposing factor in 11.1% of cases. Fusarium solani (25%) was the commonest isolate followed by Aspergillus species (19%), Curvularia species (18.5%) and Penicillium species (15.2%). Yeasts were isolated in 2.7% (n=5) cases. Conclusions: Ocular trauma was the commonest cause of fungal corneal ulcer in Assam and Fusarium solani was the commonest species responsible for it. Most of the mycotic ulcer cases come from rural areas including the tea gardens.

Atypical cutaneous lesions of Penicillium marneffei infection as a manifestation of the immune reconstitution inflammatory syndrome after highly active antiretroviral therapy.

Penicillium marneffei infections normally manifest as molluscum contagiosum like skin lesion in HIV-infected persons. We report a case with verrucous lesions over nose and face due to Penicillium marneffei infection after HAART treatment. A 28-year-old man presented, after two weeks of HAART treatment, with multiple erythematous, scaly, papules and nodules with central necrosis predominantly in face and both extremities and scrotum. Microbiological investigations confirmed the diagnosis of Penicillium marneffei infection. This is perhaps the first case report with such manifestation due to Penicillium marneffei infection.

Extended spectrum beta lactamase producing gram negative bacilli in a tertiary referral hospital of Assam--experience with two methods.

Extended Spectrum Beta Lactamases (ESBL) are enzymes produced in some gram negative bacilli that mediate resistance to extended spectrum cephalosporins. 683 clinical isolates of Escherisia coli and Klebsiella pneumoniae were studied for their capacity to produce ESBL. Isolates showing resistance to at least two of the third generation cephalosporins were studied for ESBL production by Jarlier technique and combination disc methods. Out of the 457 E. Coli and 226 Klebsiella pneumoniae isolated in Assam Medical College, 29.76% and 53.1% were resistant to two cephalosporins of which 29.41% and 29.16% strains showed production of ESBL. However, 6 (4.41%) and 16 (13.34%) strains additionally showed production of ESBL when tested with combination disc method. Though the Jarlier technique is popular, for detection of ESBL, yet false negative results warrants for alternative method. In the absence of molecular detection methods in routine clinical microbiology laboratory, combination disc method appears to be a better option.