Microbiologia Médica e Diagnóstico

American Tegumentary Leishmaniasis (ATL) is a parasitic disease caused by protozoans of the genus Leishmania and transmitted by the bite of sandflies. It is considered neglected due to its high incidence and morbidity, mainly in developing countries. The treatment for this disease besides expensive, is toxic and has limitations such as resistance, both from the parasite and the host; and invasiveness, making it not quite acceptable for some patients. There is still no available vaccine for the disease, what makes the correct diagnosis the better alternative to guide the treatment and therefore manage it. The diagnosis of suspected cases has to gather epidemiological, clinical and laboratorial data, since the disease can be misdiagnosed with other dermatological conditions. Immunological methods are commonly used for detecting and monitoring diseases, and the main techniques used in the routine for ATL are the Montenegro Skin Test, Enzyme-linked Immunosorbent Assay, Immunofluorescence Assay and Western Blot. Although these approaches have good sensitivity levels, they lack on specificity and have some limitations like crossed-reactions with other diseases caused by trypanosomatid parasites. One different serological approach, which is becoming an alternative method for the diagnosis of this disease, is Flow Cytometry; it has shown balanced levels of specificity and sensitivity and also a better accuracy when compared to other methods used in routine. Therefore, this article shows the recent advances on the diagnosis of ATL by flow cytometry and by using the findings on the literature, aims to guide researchers on what should be the focus to have a better diagnostic method for this disease.

Paratyphoid fever has been emerged as a global public health problem, especially in developing countries. There should be a consistent monitoring of the isolation throughout the countries to analyze the spectrum of the Salmonella enteric serovar Paratyphi. This study examined current isolation pattern of Salmonella Paratyphi A, B and C over a period of 6 years at National Salmonella and Escherichia Centre (NSEC), Central Research Institute (C.R.I), Kasauli. Miscellaneous suspected cultures of Salmonella had been received from various regions of India during six years span of January 2010-December 2015. These samples were characterized by biotyping as well as serotyping at NSEC situated at Central Research Institute, Kasauli. Isolates were serotyped on the basis of somatic O and phase 1 and phase 2 flagellar antigens by agglutination tests with antisera according to the Kauffmann White scheme. Out of 71 isolates of Salmonella enterica, 51 (71.830%) were Salmonella Paratyphi A, 16 (22.532%) were Salmonella Paratyphi B and 4 (5.633%) Salmonella Paratyphi C. Among total 71 samples of Salmonella Paratyphi, 11.267% were obtained from North India while 88.732% cases were from South India. 81.96% samples were isolated from blood while rest of samples were from feces, urine, pus etc. Increasing rates of antibiotic resistance among S. enterica, particularly in Salmonella Paratyphi A strains, is of concern, as Salmonella Paratyphi A infection is becoming increasingly common and is not prevented by current vaccinations. This study caters the Salmonella Paratyphi A, B, C characterization by biotyping and serotyping status in various regions of India irrespective of their co-relation to the region of isolation, source of sample isolation, types of isolates including their age, gender and season during period of 2010 to 2015 across India.

The main aim of the study was to test for the antimicrobial potency of Aloe secundiflora, Bulbine frutescens, Tagetes minuta and Vernonia lasiopus against Staphylococcus aureus. All the plants showed a pronounced antimicrobial activity against Staphylococcus aureus with Tagetes minuta being the most active at low concentrations (MIC 8.9 mg/ml; MBC 10.0 mg/ml) whereas Vernonia lasiopus showing less activity (MIC 12.2 mg/ml; MBC 14.2 mg/ml). The efficacy test was carried out using the disc diffusion method. The standard antibiotics used were ciprofloxacin (5 μg/ml) and vancomycin (3 μg/ml) showed significant antimicrobial activity by producing zones of inhibition of 22 mm and 25 mm respectively. Dimethyl sulphoxide and distilled water were used as negative control. The extracts from the plants were also screened for the presence of phytochemicals with the results showing the presence of flavonoids, alkaloids, tannins and saponins in all the extracts. The study suggested that the selected medicinal plants can be used effectively in the treatment of bacterial infection caused by Staphylococcus aureus.

In connectivity to the introduction of frequent epidemics of influenza A (H3N2, H1N1), the Influenza Surveillance Projects has monitored the burden of influenza in the outpatients through population-based surveillance. WHO is playing an active role by keeping an eye on status of seasonal as well as epidemic strains of influenza by its surveillance throughout the world. The present study offers the status of ongoing surveillance influenza at National Influenza Surveillance Centre (NISC), Central Research Institute (CRI), Kasauli for the emergence and consistency of influenza strains for current as well as over a period of three decades in Solan, Himachal Pradesh. From 1980 to 2015, various clinical centers of Himachal reported counts of influenza-like illness (fever including cough or sore throat). During these years, the respiratory specimens of 6581 patients showing influenza-like illness were collected. The incidence of visits had been calculated for influenza-like illness using the size of the patient population, and the incidence attributable to influenza was extrapolated from the proportion of patients with positive tests. Up to 2008 the egg inoculation method was used for the isolation and detection of influenza strains but in 2009 a RT-PCR equipped, fabricated BSL-3 laboratory was implanted at CRI for the isolation and detection of influenza strains. The reagents, primers and probes were supplied by NCDC, Delhi. Since 1980, 319 influenza isolates has been identified and isolated at NISC. Among these 282 were isolated by egg propagation method while 37 were processed by RT-PCR. Influenza incidence varied with age groups and by season after the pandemic of 2009 influenza A, H1N1. High levels of influenza virus circulation, especially in young Children, emphasize the need for additional efforts to increase the uptake of influenza vaccines and anti-viral. The continuous surveillance and monitoring of influenza strains is necessary to keep a check on the highly variable influenza strains to avoid the deadly incidents of influenza pandemics. In current scenario RT-PCR method is successful and appropriate method for the influenza surveillance.

The statistics on HIV in Russia in 2013 were studied and compared with the blood service data. It was found that among different contingents the minimum detection rate of HIV infection has been registered among blood donors. Maximum detection rate of HIV among men, who have sex with men, actualizes the need for a ban on their participation in blood donation. The detection rate of HIV among donors, as well as the ratio of detection of HIV among donors and other categories surveyed indicate a lack of efficiency of formation of donor contingent of supporters of a healthy lifestyle. A direct positive correlation of HIV was detected in blood donors and region population, the volume of blood processing, as well as all volumes of blood wastage due to markers of blood borne infections except the volume due to HBs-antigen.

Background and Objectives: Switch of cytokines from a T helper 1 (Th1) to a Th2 is an important factor in the progression of HIV infection to AIDS. Hence, our objectives were to analyze the levels of Th1 (IL-2, IFN-γ) and Th2 (IL-4, IL-10) cytokines and their correlation with clinical and immunological profiles in HIV/AIDS patients.
Methods: We studied 234 symptomatic HIV positive patients (case group) attending OPDs, ART clinic and medical wards of the Hospital. CD4+T-cell count was determined by flow cytometry using Fluorescent Activated Cell Sorter Count system. Quantitative determination of cytokines (Th1 subtype: IL-2, IFN-γ and Th2 subtype: IL-4, IL-10) was done by ELISA.
Results: Patients (08-68 years) had CD4+T-cell counts ranged from 16-1033 cells/μl. The median CD4+T cell count was 204.50 cells/μl and the mean was 265.48 cells/μl. The concentration of IL-2 and IFN-γ were significantly lower in case group compared to asymptomatic HIV patients (P<0.001) while the IL-4 in symptomatic HIV negative was higher than healthy controls and case group compared to asymptomatic HIV patients. Concentration of IL-10 was also higher in case group compared to asymptomatic HIV patients. A positive correlation was found between IL-4, IFN-γ, IL-2 with CD4+T-cells and a negative correlation between IL-10 and CD4+T-cells among the case group.
Conclusions: Hence a decline in type-1 cytokines (IFN-γ, IL-2) and rise in type-2 cytokines (IL-4, IL-10) was observed in symptomatic patients with HIV infection in comparison to asymptomatic HIV patients suggesting a shift from Th1 to Th2 type cytokine response.

Background: Determination of TREM-1 expression on monocytes has been investigated as a perspective diagnostic method to distinguish infectious from non-infectious etiology of the inflammation.
Objectives: To analyse the expression of the TREM-1 gene in patients of sepsis and septic shock and its relation with the severity of pathophysiological conditions. Also to describe the dynamics changes of soluble TREM- 1, procalcitonin and C-reactive protein during the course of sepsis and Correlation between their expressions and the clinical scoring system known as APACHE II scoring system.
Material and Methods: After an ethical approval, the expression of mRNA of TREM-1 in PMNs was detected in 76 critically ill patients (49 with sepsis and 27 with septic shock) were analyzed by using quantitative real-time PCR. The concentration of s. TREM-1, CRP and PCT levels were measured by an enzyme immunoassay.
Results: On the ICU admission, the septic shock group displayed higher levels of sTREM-1, PCT, APACHE II score (P<0.001) and CRP (P=0.01) than the sepsis group. Significant positive correlation (P<0.05) between APACHE II and both sTREM-1 and PCT in the prognosis of morbidity were shown. The mRNA levels of patients suffering from sepsis and septic shock were 0.54- and 0.39-fold lower compared to those of healthy subjects (1.06) respectively.
Conclusion: With regard to sepsis diagnosis and severity, sTREM-1 and PCT have positive values in prognostic assessment of the disease and may be taken as a survival-impacting risk factor. The TREM-1 gene expression levels isolated from patients with sepsis may be used as a surrogate marker for determining the severity.

Introduction: Klebsiella pneumonia is an emerging pathogen associated with multidrug resistance both in hospital and community settings. Aminoglycosides, considered to be second line drug for the treatment of such pathogens, become inactive due to acquisition of various resistance determinants by this organism.
Objective: The objective of the study was to screen the aminoglycoside resistant Klebsiella pneumonia from a tertiary referral hospital of northeast India and their transmission dynamics.
Method: A total of 177 consecutive, non-duplicate, clinical isolates of Klebsiella pneumonia were collected from patients from a period of September 2013 to February 2014. Screening for aminoglycoside resistance was performed. Transferability of aminoglycoside resistance was done by transformation assay. Genetic stability was checked by consecutive serial passage of 70 days. Incompatibility types were determined by PCR based replicon typing.
Result: Among 177 clinical isolates, 94 were screened to be resistant towards aminoglycoside group of antibiotics. The aminoglycoside resistance determinant was found to be transferable when transformants were selected in gentamicin (100 μg/ml) screen agar. Coresistance was also shown by these isolates. Gentamicin resistance was lost after 47 consecutive serial passages. F inc type (n = 17) was more predominant, followed by K/B (n = 11), Y (n = 13), I (n = 9) and P (n = 8) when plasmids were typed by PCR based replicon typing.
Conclusion: This study highlighted the transmission dynamics of aminoglycoside resistance determined which pose threat to the treatment option in hospital settings.

Methicillin resistant Staphylococcus aureus (MRSA) is a major human pathogen responsible for several life threatening conditions. MRSA have the ability to acquire resistance to several antimicrobial agents and phage therapy is one potential option to treat this pathogen. The aim of the study was to isolate and characterize bacteriophages effective against a wide range of methicillin-resistant Staphylococcus aureus (MRSA). A mixture of ten MRSA isolates was used for the isolation of phage from wastewater treatment plants. Three phages were selected for further characterization. All three phages belong to the Siphoviridae family and have long non-contractile flexible tails. The three phages showed a wide host range against S. aureus. Phages ɸSA1 and ɸSA2 were resistant to a pH range from 4-10 while ɸSA3 has a pH range from 3-11. DNA from all three phages was resistant to digestion by endonuclease enzymes such as EcoRI and AccI. There was a high degree of mosaicism among the three virulent phages and with their ancestor phages of Siphoviridae due to their non-uniform access to the common genetic pool by horizontal gene transfer and recombination. Since some of the staphylococcal toxins are phage encoded, the presence of genes for such toxins was tested by performing polymerase chain reaction and all three phages lacked genes for any of the staphylococcal toxins, including staphylococcal enterotoxins (sea, seb, sec and see), exfoliating toxins (eta and etb) and the toxic shock syndrome toxin (tst), therefore these bacteriophage are suitable candidates for future use in phage therapy against MRSA.

Pyometra, a hormonally mediated diestrual disorder is described by thickening of the uterine lining and accumulation of uterine secretions (cystic endometrial hyperplasia). These secretions leaks out to the abdomen and forms ascites, which provides environment for bacterial growth. E. coli is the common organism that migrates from the vagina into the uterus and causes acsites. As a first report, a bacterium was isolated from such an ascitic fluid of pyoderma infected Rottweiler canine breed and further the organism was identified as klebsiella sp. based on biochemical tests. Antibiogram of klebsiella sp. revealed that the organism was sensitive to ciprofloxacin, erythromycin, tetracycline, ampicillin, amikacin and gentamycin and resistant to methicillin.