Detection of Beta-Lactamases (ESBL and MBL) Producing Gram-Negative Pathogens in National Public Health Laboratory of Nepal.

Background: Bacterial resistance to antibiotics has increased in recent years. Resistance to ß-lactams in Gram-negative bacteria has been reported to be associated with extended spectrum beta-lactamases and metallo-beta-lactamases. This study was aimed at determining the distribution and antibiotic susceptibility patterns of Gram-negative pathogens producing extended spectrum beta lactamases and metallo-beta lactamases. Method and Methodology. This cross-sectional study was conducted at the National Public Health Laboratory during a period of six months. All clinical specimens were obtained and processed for the identification of Gram-negative pathogens by culture, morphological, and biochemical tests. Antibiotic susceptibility testing of the isolates was performed by the Kirby Bauer disc diffusion and the isolates were tested for ESBL and MBL by the combined disk method. Results: Out of 4266 clinical specimens, 197 (4.6%) were found to be Gram-negative bacterial isolates. 47 (23.9%) isolates were ESBL producers. The most predominant organisms were Escherichia coli (53%), Klebsiella pneumonia (23%), and Pseudomonas spp. (13%). 16 (8.2%) were positive for MBL producers, and 6(3.1%) were both ESBL and MBL producers. The MBL activity was seen in E. coli (38%), followed by Pseudomonas spp. (31%), and K. pneumoniae (19%). The ESBL producers showed a higher degree of sensitivity towards imipenem and amikacin, followed by piperacillin tazobactam. MBL producers showed sensitivity towards amikacin only. Conclusion: The prevalence of ESBL and MBL producing Gram-negative bacteria was found to be high in bacterial infections in Nepal. Routine laboratory testing for ESBL and MBL is needed in order to optimize antibiotic management and reduce the risk of spread of infections caused by ESBL and MBL producers.

Diagnosis of tuberculosis from smear-negative presumptive TB cases using Xpert MTB/Rif assay: a cross-sectional study from Nepal.

BACKGROUND: In most developing countries, smear-negative pulmonary TB (SNPT) often gets missed from the diagnosis of consideration, though it accounts 30-65% of total PTB cases, due to deficient or inaccessible molecular diagnostic modalities. METHODS: The cross-sectional study enrolled 360 patients with clinical-radiological suspicion of SNPT in Tribhuvan University Teaching Hospital (TUTH). The patient selection was done as per the algorithm of Nepal's National Tuberculosis Program (NTP) for Xpert MTB/RIF testing. Participants' demographic and clinical information were collected using a pre-tested questionnaire. The specimens were collected, processed directly for Xpert MTB/RIF test according to the manufacturer's protocol. The same samples were stained using the Ziehl-Neelsen technique then observed microscopically. Both findings were interpreted; rifampicin-resistant, if obtained, on Xpert testing was confirmed with a Line Probe Assay. RESULT: Of 360 smear-negative sputum samples analyzed, 85(23.61%) found positive while 3(0.8%) of them were rifampicin resistance. The infection was higher in males, i.e. 60(25.3%) compared to female 25(20.3%). The age group, > 45(nearly 33%) with median age 42 ± 21.5, were prone to the infection. During the study period, 4.6% (515/11048) sputum samples were reported as smear-positive in TUTH. Consequently, with Xpert MTB/RIF assay, the additional case 16.5% (n = 85/515) from smear-negative presumptive TB cases were detected. Among the most occurring clinical presentations, cough and chest pain were positively associated with SNPT. While upper lobe infiltrates (36.4%) and pleural effusion (40.4%) were the most peculiar radiological impression noted in PTB patient. 94 multi-drug resistant(MDR) suspected cases were enrolled; of total suspects, 29(30.8%) samples were rifampicin sensitive, 1(1.06%) indeterminate, 3(3.19%) rifampicin-resistant while remaining of them were negative. 2(2.2%) MDR cases were recovered from the patient with a previous history of ATT, of total 89 previously treated cases enrolled However, a single rifampicin-resistant from the new suspects. CONCLUSION: With an application of the assay, the additional cases, missed with smear microscopy, could be sought and exact incidence of the diseases could be revealed.

AmpC and extended spectrum beta-lactamases production among urinary isolates from a tertiary care hospital in Lalitpur, Nepal.

BACKGROUND: Production of AmpC and extended spectrum beta-lactamases among urinary isolates has created a serious problem to the successful management of the urinary tract infection. The main purpose of this study was to determine the rates of the extended spectrum beta-lactamase (ESBL) production and AmpC beta-lactamase (ABL) production among urinary isolates. RESULTS: Among total 564 urinary isolates, 514 (91.1%) were gram negative bacilli and 50 (8.9%) were gram positive cocci. E. coli (76.1%) was the most common bacteria isolated. Staphylococcus aureus (6.7%) was the predominant gram positive bacteria isolated. 35 (6.8%) of the 514 gram negative bacilli were ESBL producers. Similarly, 14 (2.7%) of the gram negative bacilli were ABL producers. Only one isolate was ESBL and ABL co-producer. Highest rate of susceptibility of gram negative bacteria was seen toward amikacin (97.3%) followed by imipenem (94.4%). Similarly, highest rate of susceptibility among gram positive cocci was seen toward vancomycin (100%) followed by amikacin (93.5%). CONCLUSIONS: Low rates of AmpC and extended spectrum beta-lactamases production in comparison to other previous studies were reported. On the basis of the antimicrobial susceptibility patterns of the bacteria we reported in our study, amikacin, imipenem and nitrofurantoin can be used for the preliminary treatment of urinary tract infections caused by gram negative bacteria and vancomycin and amikacin for treatment of urinary tract infections caused by gram positive bacteria.

Use of Genotype MTBDRplus Assay for Diagnosis of Multidrug-Resistant Tuberculosis in Nepal.

The main aims of this study were to study the patterns of mutations in rpoB, katG, and inhA genes in Mycobacterium tuberculosis strains isolated from patients from Nepal and to evaluate the performance of genotype MTBDRplus assay, taking conventional drug susceptibility testing as gold standard for diagnosis of MDR-TB. A total of 69 Mycobacterium tuberculosis strains isolated from 73 smear positive sputum samples from patients suspected of suffering from multidrug-resistant tuberculosis were used in our study. The drug susceptibility pattern of Mycobacterium tuberculosis isolated from these sputum specimens was determined by using genotype MTBDRplus assay taking conventional drug susceptibility testing as reference. The sensitivity and specificity of the genotype MTBDRplus assay for the detection of MDR-TB were found to be 88.7% and 100%, respectively. 88.7% of the rifampicin resistant isolates had mutations in rpoB gene. Similarly, 79.7% and 9.4% of isoniazid resistant isolates had mutations in katG and inhA genes, respectively. Genotype MTBDRplus assay was found to be very rapid and highly sensitive and specific method for diagnosis of MDR-TB and will be very helpful for early diagnosis of MDR-TB in high tuberculosis burden countries.

A hospital based surveillance of metallo-beta-lactamase producing gram negative bacteria in Nepal by imipenem-EDTA disk method.

BACKGROUND: A rising threat of the rapid spread of acquired metallo-beta-lactamases (MBLs) among major Gram-negative pathogens is a matter of public health concern worldwide. Hence, for a low income nation like Nepal, surveillance data on MBL producing clinical isolates via a cost effective technique is necessary to prevent their dissemination as well as formulation and regulation of antimicrobial stewardship policy. METHODS: The prospective study was conducted at Nepal Medical College, Kathmandu from May to October, 2014 to assess the prevalence of MBL production among ceftazidime-resistant Gram-negative rods (GNRs) isolates. The samples were processed according to standard microbiological procedure following the Manual of clinical Microbiology. Isolated GNRs were subjected to susceptibility testing against the selected panel of antibiotics by Kirby- Bauer disc diffusion method and interpretation made in conformity with the Clinical and Laboratory Standards Institute (CLSI) guidelines. Ceftazidime-resistant isolates were subjected to the detection of MBL production by imipenem-EDTA combined disc (CD) method. RESULTS: Among the Gram-negative isolates, 5.80% (21/362) were found to be MBL positive with Acinetobacter spp. showing the highest prevalence i.e. 85.71% (18/21), followed by P. aeruginosa i.e. 14.29% (3/21). None of the other cefazidime resistant gram negative bacteria tested were found to be positive for MBL production with all the positive isolates determined to be Multidrug resistant (MDR) strains. CONCLUSION: This study demonstrated a higher rate of resistance among P. aeruginosa and Acinetobacter spp. to a wide variety of antibiotic categories with an additional burden of MBL production within them, warranting a need for strict surveillance and rapid detection of MBL production among the GNRs.

Microbiological profile of corneal ulcer cases diagnosed in a tertiary care ophthalmological institute in Nepal.

BACKGROUND: Corneal ulcer, a major cause of monocular blindness in developing countries has consistently been listed as the major cause of blindness and visual disability in many of the developing nations in Asia, Africa and the Middle East, ranking second only to cataract. This study was carried out to determine the microbiological profile of corneal ulcer cases diagnosed among patients visiting Tilganga Institute of Ophthalmology (TIO), Nepal. METHODS: A total of 101 corneal scrapping samples were tested for routine culture and antibiotic susceptibility at the pathology department of TIO Nepal from April to October 2014. Microorganisms were identified by using standard microbiological procedures following the manual of American Society for Microbiology (ASM) and their antibiotic susceptibility test, performed by Kirby-Bauer disc diffusion method in conformity with the CLSI guideline. RESULTS: Out of 101 samples analyzed, 44.6% (45/101) showed positive growth with bacterial isolates i.e., 56% (25/45), more prevalent than fungus i.e., 44% (20/45). Among bacteria Streptococcus pneumoniae (31.1%, N = 14) was isolated in highest number whereas Fusarium (13.4%, N = 6) was the most common fungus species. Pseudomonas aeruginosa was the only Gram negative bacteria isolated from corneal ulcer cases. All bacterial isolates were found to be susceptible to the quinolone group of antibiotics (moxifloxacin followed by ofloxacin and ciprofloxacin). CONCLUSIONS: These findings showcase the current trend in the microbiological etiology of corneal ulcer in Nepal, which have important public health implications for the treatment as well as prevention of corneal ulceration in the developing world.

Enteric parasitic infection among HIV-infected patients visiting Tribhuvan University Teaching Hospital, Nepal.

BACKGROUND: Enteric parasitic infection among human immunodeficiency virus (HIV) patients has been a significant health problem in developing countries like Nepal. This study was undertaken to access the burden of enteric parasites among HIV patients and its association with their immune status. METHODS: A cross-sectional study, involving 112 HIV sero-positive patients was conducted in Tribhuvan University Teaching Hospital, Public Health Research Laboratory, Kathmandu, Nepal from July 2011 to June 2012. The fecal samples were processed by direct-smear technique, in both normal saline solution and 1% iodine solution as well as modified acid fast staining (Kinyoun's method) after formalin ether concentration and Sheather's sucrose flotation for the identification of enteric parasites. RESULTS: Infection with one or more parasite was seen in 33.9% (n = 38) of the cases enrolled in the study, with the parasite prevalence rate of 41.1% (n = 46). Literacy (OR = 1.9, 95% CI 0.9-4.3) and CD4 T-cell count <200 (OR = 2.5, 95% CI 1.1-5.7) were found to be associated with enteric parasite infection. Similarly, CD4 T-cell count <200 was found to be associated with opportunistic parasitic infection (OR = 3.2, 95% CI 1.2-7.8). Among opportunistic parasites, Giardia duodenalis was the most common (28.3%, n = 13) one. Multi-parasitism was observed in six patients (15.8%). CONCLUSION: Enteric parasitic infections are common in HIV-infected people. The poor immune status as indicated by low CD4 T-cell count may account for higher risk of both opportunistic and non-opportunistic enteric parasitic infection.