FROM MICROBIOLOGY TO CLINICAL MICROBIOLOGY

CAN WE MAKE IT? Dr.T.V.Rao MD
The Curriculum in Microbiology in the Indian contest remained static for several decades; the training in MD (Microbiology) under Medical Faculty remained without clinical approaches in Diagnosis and treatment. Much of Medical Microbiology remained to the Laboratory with little interest in the Hospital practice. Every Medically qualified Microbiologist spends Major years of life in a speciality without much productivity and newer innovations. Thanks to several curricular changes in efforts to make subject, as par with a Clinical Speciality. Fewer allocations from both Government and Private Institutions in funding and shortage of qualified technicians have brought the speciality to brink of deterioration. The Pandemic of AIDS awakened the Society and Doctors that spread of AIDS cannot be controlled with Laboratories, but our continuous understanding of the Society, and greater understanding of the disease with clinical knowledge. I think it is time that we should become active to make Microbiology as Clinical Microbiology with our united efforts. At present we all study in-depth Microbiological aspects dealing particularly with theoretical and little practical knowledge of medical diagnostics. It also focuses on molecular and conventional techniques for isolating, identifying and characterizing bacterial, viral, fungal and parasitic pathogens, as well as prevention and control of infectious diseases, epidemiology, and details of specimen collection, handling, examination and interpretation of results. However a little interactions with our knowledge of Medicine make greater contributions. At present curriculum is elaborate and wide without much specificities, creates greater confusion. My little experience as qualified in MD from reputed Government Institution make me to realize that I do not practice even 10% of the knowledge gained in my post-graduation, and Post graduate degrees make you a undergraduate teachers at the most a postgraduate examiner after years of our service. It is questionable how much we are productive to the society. The reasons are many clinicians go on their own way thinking antibiotics can solve all problems, which has led to growing concerns on Antibiotic multidrug, pan drug resistant strains. We can make a change in thinking with practice of few good ideals as ……… 1 Make use of suitable range of diagnostic, investigative and/or monitoring procedures when undertaking investigations, which can give optimal, care of the patients. 2 Communicate complex and technical information to patient’s, colleagues and those with limited technical knowledge in terms that facilitate understanding of issues. 3 Accept the responsibilities of the role of the scientist-trained person in relation to other health care professionals and with empathy and sensitivity to patients, and families. 4 Ensure validations of data, through use of appropriate sources of information including relevant databases and consultation with senior colleagues. 5 Use laboratory Information Technology, WHONET systems for handling, processing and storage of patient data.

CLINICAL EXPERIENTIAL LEARNING The recommended examples of clinical microbiology learning are • Prepare a portfolio of significant clinical cases reported in your hospital and be able to list clinical outcomes and main learning points. • Participate in multidisciplinary review meetings at which bacteriology and other diagnostic results are presented as part of the clinical record. • Critically appraise the internal quality control and external quality assessment of different routine bacteriology methods and draw conclusions about method performance and quality. Our Postgraduate Students should be, familiar with • The clinical impact of important bacterial and viral infections and of the appropriate clinical and laboratory investigations. • The interpretation and reporting of laboratory results in the context of important bacterial and other Microbial infections. • The partnership between the clinical microbiology laboratory and other clinical specialties in the investigation of common bacterial infections. As we are aware many specimens sent to Microbiology are sent for Histopathological and biochemical examination too, our constant touch with our colleagues makes our reports acceptable and causes no confusion among the Clinicians. • Perform and critically evaluate the results from a range of assays used to investigate viral infection including: immunoassay; agglutination; immunofluorescence; neutralization; Immmunoblotting, electrophoresis and gel diffusion. Many rapid tests we do in the laboratory are inconclusive, and may not be specific, and Microbiologists should accept the limitations and try for better and newer generation of tests, or else we become static in progress of the patients care. • Clinical practice guidelines if followed by Clinical microbiologists, have several potential benefits, including better patient care at lower costs and, when applied properly, the potential to protect health care providers from legal claims. There are several potential reasons we are failing to follow, many standard guidelines, due lack of coordination at many levels of administration, resources and funding crunch as in most of the Developing countries. Still we can do better being aware of clinical practice guidelines. It is not surprising that clinical microbiologists might not be aware of a specific aspect of clinical practice guidelines. Many of us are confused with different conflicting interpretative criteria; there are over 1,000 clinical practice guidelines in the National Guidelines Clearinghouse database, all of which make recommendations that directly affect laboratory practice. • Overcoming the challenges, remains a challenge to all upcoming Microbiologists, until the Medical fraternity realizes, Microbiology is not an armchair job as in the past but active Life Saving Clinical Specialty.

Changing role of Microbiologists – Simple Measures to Improve our Laboratories - Now the Society realises that Antibiotic Resistance is a concern to everybody’s health and a future concern for even a Healthy person, We should be proactive and prove that we are leaders to handle the situation, with even little developments in our Laboratories, will prove useful to the Medical profession. Hospital Microbiology laboratories should follow standard protocols for susceptibility testing, possible with following with CLSI guidelines and reporting on WHONET software 5.6 which also can be changed from default to the currently accepted zone sizes in reporting, Must generate and distribute Antibiograms at regular intervals (Quarterly) to the Clinicians so they can be made aware how ineffective some antibiotics have become. Hospitals to send antimicrobial susceptibility testing (AST) to standardized labs to avoid erroneous reporting of organisms and their susceptibility pattern. Pandrug-resistant Gram-negatives, carbapenem-resistant Gram-Negatives, Vancomycin- resistant Enterococcus and MRSA should be made notifiable. We can create collaborating laboratories around us and strive to improve the quality control of isolates for identification emerging trends in antibiotic resistance. Know your Antibiotics; Testing for Cephamycins in Diagnostic Laboratories - Cephamycins are a group of beta-lactam antibiotics. They are very similar to cephalosporins, and the Cephamycins are sometimes classified as cephalosporins. They are characterized by the presence of a 7-α-methoxyl group, which confers unusually high resistance to β-lactamases. Cefoxitin, the first semisynthetic Cephamycin, is resistant to almost all β-lactamases. Like cephalosporins, Cephamycins are based upon the cephem nucleus. Unlike most cephalosporins, Cephamycins are a very efficacious antibiotic against anaerobic microbes. Semisynthetic, Cephamycins are produced by Streptomyces spp.; includes cefoxitin sodium, cefmetazole and cefotetan. The significant resistance to β-lactamase displayed by the Cephamycins is reflected in the kinetics of enzyme activity (Km and Vmax) that are reported for the cephalosporins and the Cephamycins. Resistance to β-lactamase is probably one of the reasons that many cephalosporin-resistant cultures are susceptible to Cephamycins. Active against many cephalothin-resistant gram-negative bacteria; cefoxitin demonstrates a very broad spectrum that includes indole-positive Proteus and many strains of Serratia. In contrast to that of the cephalosporins, cefoxitin's spectrum of activity against anaerobic pathogens includes Bacteroides fragilis. Cefoxitin is bactericidal and almost devoid of any inoculum effect. We have to test for the susceptibility patterns for Cephamycins independent of 3rd generation Cephalosporins, and report for the utility of these drugs.

Know your Microbes - Threat of Multidrug-Resistant Gonorrhoea, -- Gonorrhoea has affected
humans for centuries and remains common Sexually transmitted Infection Worldwide, an estimated 106.1 million cases occur annually. Many patients infected with Gonococcus infections approach unqualified, even chemists and few times a family physician for a treatment, and patients may not give the proper history and will be subjected to very unscientific approaches with ineffective antibiotics. A major challenge to monitoring emerging antimicrobial resistance of N. gonorrhoea is the substantial decline in capability of laboratories to perform essential gonorrhoea culture techniques required for antibiotic susceptibility testing. Specific diagnosis of infection with N. gonorrhoea can be performed by testing endocervical, vaginal, urethral (men only), or urine specimens. Culture, nucleic acid hybridization tests, and NAATs are available for the detection of genitourinary infection with N. gonorrhoea. Since antibiotics were first used for treatment of

gonorrhoea on empirical basis, N. gonorrhoea has progressively developed resistance to the antibiotic drugs prescribed to treat it: sulfonilamides, penicillin, tetracycline, and ciprofloxacin. When bacteria become resistant to an antibiotic, they no longer can be killed by that medicines prescribed; there is no reliable technology that allows for antibiotic susceptibility testing from nonculture specimens; Antimicrobial resistance in Neisseria gonorrhoea may arise by selection of resistant mutants from endogenous flora produced by spontaneous chromosomal mutations, or it can be acquired from other bacteria by plasmid-mediated transfer or DNA transformation and recombination. Plasmid-mediated resistance to tetracycline and penicillin, and chromosomally mediated resistance to tetracycline, penicillin, and spectinomycin can occur. As of recently, increased resistance to fluoroquinolones has also been observed. This can happen by alteration of drug targets, restriction of access to bacterial targets, or both; thus, fluoroquinolone potency is determined by its ability to reach and act on their target enzymes, DNA gyrase and topoisomerase IV, which are both required for bacterial DNA replication . However we are rarely getting a specimen for culturing Gonococcus in majority of our laboratories. In those who fail initial treatment, culture should be done to determine sensitivity to antibiotics. Clinicians many times take decision to treat the disease with accumulated experience, Fluoroquinolones such as ciprofloxacin or Ofloxacin are no longer recommended as first-line therapies for gonococcal infections. Patients infected with N. gonorrhoea frequently are coinfected with C. trachomatis; this finding has led to the recommendation that patients treated for gonococcal infection also be treated routinely with a regimen that is effective against uncomplicated genital C. trachomatis infection .Therapy for gonorrhoea is often given before the susceptibility of the infecting organism is known, the Centres for Disease Control and Prevention recommends that uncomplicated gonorrhoea be treated only with the antibiotic ceftriaxone — given as an injection — in combination with either azithromycin or doxycycline — two antibiotics that are taken orally. Many times the partner of the patient is missed for diagnosis and treatment, one should remember the partner also should undergo testing and treatment for gonorrhoea, even if he or she has no signs or symptoms. Partner receives the same treatment; you give to the infected partner. Even if you've been treated for gonorrhoea, one can be reinfected if his/her partner isn't treated. Untreated gonococcus infection can lead to pelvic inflammatory disease, ectopic pregnancy, and infertility in women and can facilitate transmission of human immunodeficiency virus. Childhood blindness still affects infants born to mothers infected with gonorrhoea, particularly in resource-limited countries. NIAID is studying new ways to treat cephalosporin-resistant infections by using existing antibiotic therapies in combination (i.e., gentamicin and azithromycin vs. Gemifloxacin and azithromycin).

Know your Microbes - Challenges in treating Enterococcus Infections --Enterococci
species can cause a variety of infections, including urinary tract infections, bacteraemia, endocarditis, and meningitis. The combination of high-level resistance to ampicillin, Vancomycin, and aminoglycosides is now fairly common among hospital-acquired Enterococcus faecium has a major impact on therapeutic options. Compared with most streptococci, enterococci are relatively resistant to penicillin and ampicillin; even when these cell wall-active agents inhibit enterococci, they often do not kill them, and Vancomycin is even less bactericidal. And imipenem has some activity against E. faecalis. Cell wall-active agents with limited or no activity against enterococci include nefcillin, oxacillin, Ticarcillin, Ertapenem, most cephalosporins, and aztreonam. Enterococci are also relatively impermeable to aminoglycosides, and the serum concentrations of aminoglycosides required for bactericidal activity are much higher than can be achieved safely in humans. The

simultaneous use of a cell wall-active agent is needed to raise the permeability of the cell so that an intracellular bactericidal aminoglycoside concentration can be achieved without excessive toxicity. Enterococcus isolates are usually tested for susceptibility to ampicillin, penicillin, and Vancomycin. Prior to using penicillin or ampicillin for endocarditis or other life threatening enterococcus infections, such as meningitis, the isolate should also be screened for beta-lactamase production with nitrocefin, a chromogenic cephalosporin, even if ampicillin susceptible. Although found only rarely, the presence of beta-lactamase confers resistance to penicillin and ampicillin when large numbers of organisms are present (eg, endocarditis vegetation’s), even though the organism may test susceptible using standard laboratory inoculum. Isolates from life threatening infections should also be tested for high-level resistance to gentamicin and streptomycin. If the organism is reported as susceptible to high levels of an aminoglycoside ("SYN-S" indicates "susceptible to synergism"), then it is assumed that synergism will be achieved with that aminoglycoside. Strains that are resistant to high levels of gentamicin are resistant to synergism with tobramycin, netilmicin, and amikacin as well as gentamicin, but some of these strains lack high-level resistance to streptomycin and will demonstrate synergism with that agent. Newer antibiotics (eg, quinupristin-dalfopristin, linezolid, Daptomycin, Tigecycline) with activity against many VRE strains have improved this situation, but resistance to these agents has already been described. A mutation (G2576U) in the domain V of the 23S rRNA is responsible for linezolid resistance, whereas resistance to quinupristindalfopristin may be the result of several mechanisms: modification of enzymes, active efflux, and target modification. Resistance of E faecalis and E faecium to Daptomycin, a newer cyclic lipopeptide antibiotic that acts on the bacterial cell membrane, has also been reported. Think before treating and suggesting treatment for Enterococcus infection.

Know your Microbe Community-Associated MRSA (CA-MRSA)

Refers to an MRSA

infection with onset in the community in an individual lacking established MRSA risk factors, such as recent hospitalization, surgery, residence in a long-term care facility, receipt of dialysis, or presence of invasive medical devices. CA-MRSA isolates commonly possess genes for the Panton-Valentine Leukocidin (PVL) toxin, rarely identified in HA-MRSA isolates. Presence of PVL genes in S.aureus isolates has been associated with primary skin infections. From a clinical management standpoint, awareness of local resistance patterns for pathogens in the differential diagnosis of specific clinical syndromes is more important than formally categorizing possible MRSA infections as CA-MRSA or HA-MRSA; Factors common to these settings that facilitate the spread of infection include crowding, frequent skin-to-skin contact between individuals, participation in activities that result in compromised skin surfaces, sharing of personal items that may become contaminated with wound drainage, and challenges in maintaining personal cleanliness and hygiene. Community outbreaks have been reported in sports teams, child care attendees, prison inmates, and diverse populations where habitation is relatively concentrated. Isolates obtained from these patients with MRSA infections described as CA-MRSA based on epidemiologic criteria have been noted to possess bacteriologic characteristics distinct from those of isolates from patients meeting epidemiologic criteria for HA-MRSA, although this situation is evolving. CA-MRSA isolates tend to be resistant to fewer antimicrobial classes, possess different toxin genes, and carry a different type of the gene complex. Unlike HA-MRSA isolates, which are usually resistant in vitro to multiple classes of antimicrobial agents, many CA-MRSA isolates to date have been resistant only to beta-lactams (the antimicrobial class that includes penicillins and cephalosporins) and macrolides / azalides (e.g., erythromycin, clarithromycin, azithromycin). However, resistance to other classes of antimicrobial

agents, such as fluoroquinolones and tetracycline’s, occurs and may be increasing in prevalence. Most CA-MRSA isolates to date have been susceptible to trimethoprim-Sulphmethoxazole (TMP/SMX), gentamicin, tetracycline, and clindamycin; although some S. aureus isolates that appear erythromycin-resistant and clindamycin-susceptible by routine susceptibility testing by disc diffusion methods exhibit in vitro resistance to clindamycin during therapy (“inducible resistance”) called the D-zone test. S. aureus strains with the inducible resistance phenotype, termed “inducible macrolidelincosamide-streptogramin B resistance” (iMLS), have a high rate of mutation to constitutive clindamycin resistance, a trait which would confer a selective advantage during clindamycin therapy. Although erythromycin is used to induce clindamycin resistance in the D-zone test, pre-treatment or co-treatment with erythromycin is not needed for iMLS strains to express clindamycin resistance in vivo during a course of therapy. If empiric Clindamycin therapy has been initiated and inducible clindamycin resistance is detected, response to therapy should be assessed. Clinicians should consider changing to another agent if response to therapy has been unsatisfactory and should monitor the patient closely to assure resolution of the infection, if clindamycin therapy is continued. The problem to identify CA-MRSA continues to be difficult in most of Laboratories as we routinely do not do Genotyping for differentiating between HA-MRSA and CA-MRSA. An optimal clinical filling of the request from with all relevant details can be able to give some clues to identify at least few with Antibiogram patterns. Too much dependence of empirical treatment by Clinicians can lead to increased morbidity and Mortality as CA-MRSA can produce life threating complications if not effectively treated. Only vigilant prevention and implementation of the most current treatment protocols will provide an increased margin of safety.

Know your Antibiotics - Aztreonam is the first member of a new class of beta-lactam antibiotics,
the Monobactams. It was originally isolated from Chromobacterium violaceum. It is a synthetic bactericidal antibiotic. The Monobactams, having a unique monocyclic beta-lactam nucleus, are structurally different from other beta-lactam antibiotics (eg, penicillins, cephalosporins, Cephamycins). The sulfonic acid substituent in the 1-position of the ring activates the beta-lactam moiety; an aminothiazolyl oxime side chain in the 3-position and a methyl group in the 4-position confer the specific antibacterial spectrum and beta-lactamase stability. It is resistant to some betalactamases, but is inactivated by extended-spectrum beta-lactamases. Aztreonam has strong activity against susceptible gram-negative bacteria, including Pseudomonas aeruginosa. It has no useful activity against gram-positive bacteria or anaerobes. It is known to be effective against a wide range of laboratory isolates of bacteria including Citrobacter, Enterobacter, E. coli, Haemophilus, Klebsiella, Proteus, and Serratia species. In 2010 the Clinical and Laboratory Standards Institute (CLSI) lowered the susceptibility breakpoints of some cephalosporins and aztreonam for Enterobacteriaceae and eliminated the need to perform screening for extended-spectrum β-lactamases (ESBLs) and confirmatory tests. Widespread resistance in Enterobacteriaceae and Pseudomonas aeruginosa to cephalosporin and Monobactam antibiotics due to extended -spectrum beta-lactamases has resulted in the need for reassessment of the interpretative criteria (breakpoints) established for these agents over 2 decades ago. Breakpoints need to be revised due to changing resistance mechanisms and bacterial population distributions, changing science leading to a better understanding of the pharmacologic determinants of clinical response, and adoption of “best practices” by clinicians. The breakpoints were revised in order to better represent the effect these agents might have when they are used to treat infections caused by contemporary isolates with currently recommended dosage regimens. Knowledge gained from ESBL-producing organisms played

a major role. From Clinical point of view based on therapeutic trials have shown aztreonam to be effective in Gram-negative infections including complicated infections of the urinary tract, in lower respiratory tract infections and in gynaecological and obstetric, intra-abdominal, joint and bone, skin and soft tissue infections, uncomplicated gonorrhoea and septicaemia. In comparisons with other antibiotics, aztreonam has been at least as effective as or more effective than Cefamandole in urinary tract infections and similar in efficacy to tobramycin or gentamicin. All the Diagnostic laboratories should not forget to test the resistance and sensitivity pattern of aztreonam as it is an equally effective Antibiotic like several 3rd Generation Cephalosporins.

Know your Antibiotics Clindamycin facts to Know – Clindamycin continues to be less used more
feared Antibiotic, in the daily practice. Clindamycin is a semisynthetic derivative of lincomycin, a natural antibiotic produced by the actinobacterium Streptomyces lincolnensis. It is obtained by It is usually used to treat infections with anaerobic bacteria, but can also be used to treat some protozoal diseases, such as malaria. Community-acquired MRSA isolates are often susceptible to several non– b-lactam drug classes, although they are usually not susceptible to macrolides. Several newer antimicrobial agents and a few older agents are available for treatment of systemic Staphylococcal infections, but use may be limited by the relatively high cost of these agents or the need for parenteral administration. Inexpensive oral agents for treatment of localized, community-acquired MRSA infection include clindamycin, trimethoprim-Sulphmethoxazole, and newer tetracycline’s. Clindamycin is used primarily to treat anaerobic infections caused by susceptible anaerobic bacteria, including dental infections, and infections of the respiratory tract, skin, and soft tissue, and peritonitis. In patients with hypersensitivity to penicillins, clindamycin may be used to treat infections caused by susceptible aerobic bacteria. It is also used to treat bone and joint infections, particularly those caused by Staphylococcus aureus. Topical application of clindamycin phosphate can be used to treat mild to moderate acne. It is most effective against infections involving the following types of organisms: Aerobic Gram-positive cocci, including some members of the Staphylococcus and Streptococcus (e.g. pneumococcus) genera, but not enterococci, Anaerobic, Gram-negative bacteria, including some Bacteroides, Fusobacterium, and Prevotella, although resistance is increasing in Bacteroides fragilis. Clindamycin is commonly used in the treatment of erythromycin resistant Staphylococcus aureus causing skin and soft tissue infections. In vitro routine tests for clindamycin susceptibility may fail to detect inducible clindamycin resistance due to erm genes resulting in treatment failures, thus necessitating the need to detect such resistance by a simple D test on routine basis. Why we do D test for Clindamycin susceptibility -When testing a Gram-positive bacteria for sensitivity to clindamycin, it is common to perform a "D-Test" to determine if there is a macrolideresistant subpopulation of bacteria present. This test is necessary because some bacteria express a phenotype known as MLSB, in which susceptibility tests will indicate the bacteria are susceptible to clindamycin, but in vitro the pathogen displays inducible resistance.

How to perform D test. In this test, an agar plate is inoculated with the bacteria in question and
two drug-impregnated disks (one with erythromycin, one with clindamycin) are placed 15–20 mm apart on the plate. If the area of inhibition around the Clindamycin disk is "D" shaped, the test result is positive and clindamycin should not be reported as sensitive due to the possibility of resistant pathogens and therapy failure. If the area of inhibition around the clindamycin disk is circular, the test result is negative and clindamycin can be reported as sensitive. The Physicians are warned

clindamycin, may cause overgrowth of dangerous bacteria like C.difficle in the large intestine. This may cause mild diarrhoea or may cause a life-threatening condition called colitis. Clostridium difficle associated diarrhoea (CDAD) has been reported with use of nearly all antibacterial agents, including Clindamycin and may range in severity from mild diarrhoea to fatal colitis. Treatment with antibacterial agents alters the normal flora of the colon leading to overgrowth of C.difficle. Clindamycin is more likely to cause this type of infection than many other antibiotics, so it should only be used to treat serious infections and patients are asked report adverse effects to the concerned Physicians. Why all are afraid of use of Clindamycin as it causes overgrowth of C.difficile which produces toxins A and B which contribute to the development of CDAD. Hyper toxin producing strains of C. difficile cause increased morbidity and mortality, as these infections can be refractory to antimicrobial therapy and may require colectomy. CDAD must be considered in all patients who present with diarrhoea following antibiotic use. Careful medical history is necessary since CDAD has been reported to occur over two months after the administration of antibacterial agents.

Note – The above published articles are contributed to various WEB resources for benefit of Young
Generation of Microbiologists Dr.T.V.Rao MD, Professor of Microbiology. Travancore Medical College, Kollam, Kerala India Email doctortvrao@gmail.com

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