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ournal of Infection and Public Health (2015) 8, 513—525

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stablishing molecular microbiologyacilities in developing countries

alman S. Ahmeda,b,∗, Emine Alpa,b,ysegul Ulu-Kilica, Mehmet Doganaya

Department of Infectious Diseases, Faculty of Medicine, Erciyes University, Kayseri,urkeyDepartment of Molecular Microbiology, Genome and Stem Cell Center,rciyes Univeristy, Kayseri, Turkey

eceived 26 November 2013; received in revised form 10 March 2015; accepted 3 April 2015

KEYWORDSDeveloping country;Molecular technique;Molecular microbiologylaboratory

Summary Microbiology laboratories play an important role in epidemiology andinfection control programs. Within microbiology laboratories, molecular microbiol-ogy techniques have revolutionized the identification and surveillance of infectiousdiseases. The combination of excellent sensitivity, specificity, low contamination lev-els and speed has made molecular techniques appealing methods for the diagnosisof many infectious diseases. In a well-equipped microbiology laboratory, the facilitydesignated for molecular techniques remains indiscrete. However, in most develop-ing countries, poor infrastructure and laboratory mismanagement have precipitatedhazardous consequences. The establishment of a molecular microbiology facilitywithin a microbiology laboratory remains fragmented. A high-quality laboratoryshould include both conventional microbiology methods and molecular microbiologytechniques for exceptional performance. Furthermore, it should include appropriatelaboratory administration, a well-designed facility, laboratory procedure standard-ization, a waste management system, a code of practice, equipment installation andlaboratory personnel training. This manuscript lays out fundamental issues that need

to be addressed when establishing a molecular microbiology facility in developingcountries.© 2015 King Saud Bin Abdulaziz University for Health Sciences. Published by ElsevierLimited. All rights reserved.

∗ Corresponding author at: Department of Infectious Diseases and8039 Kayseri, Turkey. Tel.: +90 531 381 9526.

E-mail address: biosheffield@gmail.com (S.S. Ahmed).

ttp://dx.doi.org/10.1016/j.jiph.2015.04.029876-0341/© 2015 King Saud Bin Abdulaziz University for Health Scie

Clinical Microbiology, Faculty of Medicine, Erciyes University,

nces. Published by Elsevier Limited. All rights reserved.

514 S.S. Ahmed et al.

Contents

Introduction...................................................................................................514The role of molecular techniques in clinical microbiology and infection control programs.....................515New molecular technologies .................................................................................. 516

Real-time droplet PCR .................................................................................... 516Mass spectrometry........................................................................................519Nucleic acid sequencing .................................................................................. 519Microbial volatile organic compound detection ........................................................... 519Raman spectroscopic analysis.............................................................................520Nanoparticles.............................................................................................520

Laboratory facilities in developing countries .................................................................. 520Laboratory management ...................................................................................... 520Laboratory networking systems................................................................................521Laboratory biosafety and risk assessment ..................................................................... 521Laboratory accreditation......................................................................................521Laboratory design ............................................................................................. 521Laboratory equipment.........................................................................................522Laboratory practice ........................................................................................... 523Laboratory personnel requirement ............................................................................ 523Waste management ........................................................................................... 523Prospective instructions.......................................................................................524Funding ....................................................................................................... 524Competing interests...........................................................................................524Ethical approval...............................................................................................524References .................................................................................................... 524

Introduction

Microbiology laboratories play an important role inthe management of infectious diseases in termsof national or global epidemiology. In the twenty-first century, molecular microbiology techniques forepidemiology and infection control programs havebecome more demanding. Molecular techniquesin high-quality clinical microbiological laboratoriesare routinely used to make clinical decisions on thebasis of how and when to treat patients. Molecu-lar techniques are also useful for monitoring theeffectiveness of therapeutic regimes and identi-fying potential resistant strains that may impacta long term treatment program. The manage-ment and control of infectious diseases requiresaccurate diagnosis and knowledge of pathogendistributions. The inclusion of molecular typingtechniques for almost all infectious disease agentswithin a clinical microbiology laboratory providesa detailed assessment of any given strain’s inter-relationships, and thus can identify the sourceof the organism [1]. Almost all epidemiologi-cal studies depend on molecular techniques for

uncontrollable rates of infection and mortality[2,3]. In some resource-limited countries, sentinelhospitals lack even basic microbiology laboratoryfacilities [4]. In other regions, laboratories haveall of the requisite reagents and instruments butlack skilled staff. Other laboratories can amplifyDNA but are unable to report the necessary resultsin time. Furthermore, many laboratories neglectaccreditation, quality assurance and quality controlprograms. For these reasons, infectious diseasesin most developing countries remain unknownand uncharacterized [5]. The Centers for DiseaseControl (CDC) has initiated various strategies indeveloping countries that require clinical micro-biology laboratories to provide accountable dataas part of an active surveillance of infectiousdiseases. However, clinical microbiology labora-tories have not been recognized as a priorityby government bodies in developing countries.As mentioned above, part of the problem isassociated with a lack of skilled laboratory per-sonnel and prohibitive laboratory maintenancecosts where resources are available but inappropri-ately used [6]. This manuscript discusses the role

the identification, characterization and genetictyping of microbes. Nevertheless, the lack of infra-structure for infectious disease management indeveloping countries has been blamed for the

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stablishing molecular microbiology facilities in dev

he role of molecular techniques inlinical microbiology and infectionontrol programs

onventionally, the function of a clinical microbiol-gy laboratory is to identify various pathogens usingirect examination of microbes and culture-basedethods. At present, nucleic acid amplification

nd DNA probes are useful for the characteriza-ion of microbes because cultured-based methodsre insufficient, expensive and tedious. Typi-ally, nonamplified DNA probe-based methods areuitable for in situ hybridization of organismshen the location and distribution are ascer-

ained, including slow growing microbes such aseisseria gonorrhoeae and Mycobacterium tuber-ulosis. Pathogenic dimorphic fungi have also beenetected radiometrically. Many DNA probe methodsre commercially available for the identificationf various microbial species, such as Chlamydiarachomatis, Legionella pneumophila, Trichomonasaginalis, Candida species, and human papillo-avirus (HPV). In addition to the benefit of the

onfirmation of culture methods, DNA probes areseful for identifying enterotoxin-producing strainsf Escherichia coli because these strains are bio-hemically similar to other non-enterotoxigenic. coli. Although traditional DNA probe methodsave a lower sensitivity than nucleic acid amplifi-ation techniques, DNA probes are useful for theirect identification of clinical specimens in cer-ain situations. For example, DNA probes are usedo identify group A streptococci in strep throatnfections and C. trachomatis or HPV in cervicalnfections. Furthermore, the high level sensitiv-ty of nucleic acid amplification makes it an idealhoice for the direct detection of specimens. How-ver, expensive molecular procedures should noteplace cost-effective conventional methods thatre rapid, sensitive and candid. For instance, these of shell vials and monoclonal antibodies forhe diagnosis of cytomegalovirus viremia in anmmunosuppressed bone marrow transplant recip-ent is an excellent method with high specificitynd sensitivity and results that are available within6 hours. Thus, it would be unfavorable to disre-ard this method and replace it with costlier nucleiccid amplification methods without additional doc-mentation of the substantial benefits of the latterethods.When considering the correct molecular method

o be used in the laboratory, careful and firmecision-making should be directed toward theotential benefits of modern methodology. Deci-ions should not be made based solely on

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he cost-effectiveness of the molecular method.nstead, the impact of the method on the clini-al practice and patient management should alsoe considered. For instance, direct detection ofethicillin-resistant Staphylococcus aureus and M.

uberculosis in a patient’s specimens would resultn quicker patient isolation, better targeted ther-py and overall cost savings. In other situations,icrobes can be extremely fastidious, difficult to

ulture and hazardous to laboratory staff. Theseicrobial samples are often sent to referral cen-

ers; however, fragile microbes could lose theiriability, become overgrown or even become con-aminated during the transferal process. Often, aample with a limited volume submitted to theaboratory becomes impossible to culture. In suchases, accurate usage of the sample is needed toulture all pathogens. In many resource limitedountries, biosafety level 3 laboratories may not bevailable or may be economically inappropriate toulture pathogenic microbes [7]. Molecular tech-iques have resolved many of these problems bydentifying microbes more rapidly and accuratelyhan conventional techniques. For instance, morehan 100 genotypes of HPV have been found usingNA sequence heterogeneity as the basis for inves-igation.

It is important to remember that despiteheir sensitivity, accuracy and speed, nucleic acidmplification methods cannot totally replace con-entional culture and serologic methods becauseucleic acid amplification and conventional meth-ds produce different results. Nucleic acid ampli-cation methods detect the presence of DNA orNA in a particular specimen, but they cannotemonstrate the viability of an organism or deter-ine whether the organism is part of the infectiousrocess. Furthermore, nucleic acid amplificationethods are prone to errors, and large amounts of

mplified products can lead to momentous cross-ontamination and cause false positive results.onventional methods such as culture-based tech-iques validate the viability of an organism, anduantification of the antibody titer strongly sug-ests its involvement as part of the infectiousrocess. Commercial DNA probes have broughtolecular techniques into the field of clinicalicrobiology and have been used for several years.

oday, most of the procedures have been stan-ardized and are frequently simpler than targetmplification assays. Additionally, the advent ofhe synthetic short oligonucleotide DNA probe has

educed the time required for hybridization assays.he niche for DNA probe technology in clinicalicrobiology appears to be in situ hybridization

or tissue specimens and culture conformation of

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slow-growing microbes. Although DNA probes aremore expensive than other biochemical tests, theirrapid turnaround time can be translated into overallsavings by providing a timely diagnosis. Using stan-dardized methodology, DNA probes can broaden thevarious types of pathogens that a microbiology lab-oratory can routinely identify. The three differentformats commonly used today are solution-phasehybridization, in situ hybridization and solid-phasehybridization. At present, the nucleic acid ampli-fication technique is routinely used in numerousmicrobiology laboratories. This technique improvesthe sensitivity of assays based on nucleic acids andcan simplify assays by automating and incorporatingnon-radioactive detection formats. The technologybehind the nucleic acid amplification method isdiverse and in constant flux. There are three dif-ferent categories: (i) target amplification by PCR,which relies on self-sustaining sequence replica-tion or strand disruption amplification; (ii) probeamplification including a ligase chain reaction; and(iii) signal amplification. All of these techniquesare currently used to varying degrees, and manyhave been cleared by the American Food and DrugAdministration (FDA). A detailed account of all ofthe molecular methods used in clinical microbiologylaboratories is beyond the scope of this manuscript.However, some commercially available molecularassays for various infectious agents are discussed inTable 1.

Molecular typing techniques have greatly facil-itated epidemiologists and infection control spe-cialists. The main purpose of an infection controlprogram is to identify possible infection problemsand monitor infection trends. Infection control pro-grams depend on cooperation from microbiologylaboratories, which must provide easy access tohigh-quality data and guide the use of resourcesfor epidemiological purposes. Furthermore, labo-ratory findings are used to support epidemiologicevidence of the spread of common microbes anddetermine the modes of transmission [8]. Molecu-lar typing techniques have been successfully used inmicrobiology laboratories for the epidemic surveil-lance of various pathogens [9]. Several moleculartyping methods have been proposed, such as pulsefield gel electrophoresis (PFGE), ribotyping andnucleic acid amplification-based typing schemes.Currently, pulse field gel electrophoresis is widelyused for the molecular typing of various bacte-rial species. This technique allows investigatorsto separate larger pieces of DNA using restriction

enzymes and has been used to characterize bac-terial species from different geographical regions.This technique has better discriminating powerthan other typing schemes, such as ribotyping and

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ulti locus sequence typing based on DNA amplifi-ation reactions. The results can be visualized with

dendrogram generated by matrix pair differencesetween the electrophoretic types. Despite PFGEeing considered by analogy as a ‘gold standard’ byany investigators, many strains cannot be typedue to degradation of the DNA in the gel. More-ver, the apparatus and the specialized chemicalsre expensive, time consuming and require skilledechnicians. PFGE has a lower resolution powerhan multi locus sequence typing (MLST), which is

nucleic acid amplification-based method, and asuch it is widely accepted as ‘the typing successf the decade’ because it can be applied to bacte-ia and other microbes. This technique can be usedo study the evolutionary relationships betweenacteria due to its high discriminatory power. MLSTs portable, and unlike PFGE its results can be use-ully shared between laboratories. A huge numberf microbes have been studied under these twochemes [10]. Moreover, MLST requires a normalhermocycler for amplification that can also be usedor many other direct detection assays and for thedentification of various genes, while MLST requiresn expensive nucleic acid sequencing apparatus.owever, post-amplification products can also beequenced using commercially available sequenc-ng apparatuses by paying for just the cost ofhe reaction. To date, various molecular typingechniques have been used to map various epi-emiological traits. However, the selection of theseechniques depends on the aim and objectives ofhe particular epidemiological study. This is espe-ially true in developing countries or regions whereesources are limited and skilled personnel are inemand.

ew molecular technologies

urrently, the application of nucleic acid amplifi-ation and other molecular platforms is limited toarge laboratories and referral centers. The abil-ty to use these techniques efficiently is anothermportant factor that determines how large a rolehese techniques will play in an average microbiol-gy laboratory. There is a growing need for newerechnologies that are rapid, cost effective and suit-ble for low resource countries.

eal-time droplet PCR

ecause PCR requires thermal cycling, it is typicallyerformed in a bench top unit. This process is slow,xpensive, error-prone due to contamination and

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Table 1 FDA approved commercially available molecular assays.

Organism Specimen Assay Method Target Sensitivity (%) Specificity (%)

Human papillomavirus (HPV)

Cervical cytologyspecimen

Digene HC2 HPVDNA test

Hybridizationprotection assayusing microplatechemiluminesencehybrid capture ofDNA—RNA hybrid.

RNA probe cocktail for13 h types

93.0 61.1

Chlamydiatrachomatis

Endocervical andvaginal swabs(female);urethral (males)swabs

APTIMA Assay forChlamydiatrachomatis

Transcriptionmediatedamplification

23S rRNA 95.6 98.8

Neisseriagonorrhoeae

Cervical andvaginal swabs(female);urethral (males)swabs

AMPLICOR CT-NGfor NG

PCR M.NgoPII putativemethyl transferasegene of NG

95.9—96.5 98.7—97.3

Human immuno-deficiencyvirus-1 (HIV-1)

Plasma separatedfrom bloodcollected usingEDTA

COBASAmpliPrep/COBASTaqMan HIV-1 testand COBASAmpliPrep/COBASTaqMan

Real-time reversetranscriptase-PCR

Gag gene 48—10,000,000 copies/mL (HIV-1 group M)

Herpes simplexvirus (HSV)

Vaginal lesionswab

MultiCode RTxHSV 1&2 Kit

Real-time PCR usingisoC:isoG syntheticDNA base pairtechnology

Glycoprotein genesegment of HSV-1 andHSV-2

HSV-1, 92.4HSV-2, 95.2

HSV-1, 98.3HSV-2, 93.6

MRSA/SA(screening,surveillance)

Lesion swab fromskin/soft tissue

Xpert MRSA/SAand GeneXpertSystem

Real-time PCR Staphylococcal proteinA (spa), the gene forMecA-mediatedoxacillin resistance(mecA), and SCCmecinserted in the SAchromosomal attB site

MRSA positivewith culture 93.8SA positive withculture 95.7

MRSA negativewith culture 97.3SA negative withculture 89.5

Clostridiumdifficile

Stool sample Xpert C. difficileand GeneXpert

Real-time PCR C. difficile tcdB 93.5 94.0

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Table 1 (Continued )

Organism Specimen Assay Method Target Sensitivity (%) Specificity (%)

Enterococcus Rectal swabs Xpert vanA andGeneXpertSystem

Real-time PCR Gene sequences forVanA-encodedresistance tovancomycin-teicoplanin

98.0 81.1—90.0

Organismscausing sepsis:yeast

Smears madedirectly fromyeast-positiveblood cultures

Candida albicansPNA FISH

PNA FISHspecies-specificprobes

16S rRNA of C. albicans 100 100

Group AStreptococcus

Throat swabs GAS Direct Test Hybridizationprotection assay

rRNA 91.7 99.3

Mycobacteria Growth fromsolid media orbroth

AccuProbe M.avium

Hybridizationprotection assay

rRNA 99.3 100

AccuProbeM.intracellula-re

100 100

Respiratory viruspanel: influenzaA and B and RSV

Nasopharyngealswab, culturedclinicalspecimens

VerigeneRespiratory VirusNucleic Acid Test

Multiplex RT-PCR Influenza A matrix gene Influenza A, 99.2 Influenza A, 90.1Influenza B NS andmatrix genes

Influenza B, 96.8 Influenza B, 98.5

L and F genes of RSV RSV, 89.8 RSV, 91.5

Enterovirus CSF Xpert EV andGeneXpertSystem

Real-time PCR Consensus region ofenterovirus, 5′ UTRbetween nucleotides452 and 596

96.3—100 97.0—97.2

Mycobacteriumtuberculosis

Sputum andbronchialspecimens

AMPLIFIED MTDTest

Transcription-mediatedamplification,hybridizationprotection assay

Mycobacterial 16S rRNA 96.9 100

Hepatitis B Virusquantitative

EDTA plasma orserum

COBAS TaqManHBV Test

Real-time PCR Core—precore region ofthe HBV genome;primer pairs togenotypes A—G of HBVand the precoremutation

20—170,000,000 IU/mL

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equires a large amount of sample to perform theeactions. Utilizing the concept of both spatial andemporal microfluidic PCR, droplet-based microflu-dic PCR formats are emerging as alternatives toingle-phase PCR. Droplet PCR formats have manydvantages, such as low reagent usage, fasterhermal cycling and limited interactions of DNAnd polymerase with the channel walls. Carryoverontamination can be reduced using these for-ats, resulting in high throughput and sensitivity.roplets are discrete small volumes that can bedapted for parallel amplification in small concen-rations. There are a number of pressure-drivenow systems that use microdroplets, nanodroplets,nd picodroplets to perform both PCR and reverseranscriptase PCR. Many commercially availableroducts based on real-time droplet PCR have var-ous applications, such as absolute quantification,are gene identification, genomic alterations, andingle cell analysis. A few studies have reported these of droplet PCR for the diagnosis of infectiousisease agents such as C. trachomatis (73.3% sen-itivity) and influenza subtypes (96% sensitivity and00% specificity). This technique can be adoptedn small clinics and average laboratories [11,12],specially in resource limited countries.

ass spectrometry

ass spectrometry (MS) was recently introduceds a new diagnostic technique in clinical micro-iology with the first successful application inetection and sequence-based identification ofCR products. Based on MS, matrix-assisted laseresorption/ionization (MALDI) coupled with timef light (TOF) allows the analysis of variousiomolecules in microbes. This method is veryffective and requires less intense sample prepa-ation because the matrix is less susceptible tonterference from salts and detergents. MALDI-TOFs an extremely useful technique for the identi-cation of many microbial species because theajor mass profile is generated from ribosomal pro-

eins that align perfectly with current taxonomiclassifications. For epidemiological typing studiesf bacterial isolates, mass peaks that are spe-ific to a given strain can easily be identified andsed to develop a binary typing system. Further-ore, substantial resistance mechanisms can alsoe identified. Cellular modifications under the influ-nce of antibiotics can also be detected, as wasecently demonstrated by the interaction between

andida albicans cells and fluconazole and Candidand Aspergillus species and caspofungin. Addition-lly, MS can be used to identify PCR productserived from resistance genes. The iPLEX MassArray

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eveloped by Sequenom is a very good examplef this extensible technology. However, there areany opposing issue with this diagnostic MST. The

ssays typically require 104—105 cells, which arenfrequently detected in patient specimens exceptrinary tract infections. Moreover, the equipments very expensive and needs expensive maintenance13].

ucleic acid sequencing

NA sequencing is already used in many diagnostic,yping and research studies and is particularly use-ul for bacterial identification based on 16S rRNAequences. Previously, this method was employedo identify short nucleotide sequences of PCR-mplified products. However, with advances inechnology it now is able to identify complexenome mixtures of bacteria in a process calledmetagenomics’ and can generate complete cat-logues of genomic components of the bacterialpecies present in a variety of clinical specimens.lthough at present, it remains unclear whetherhis technology is suitable for routine usage inicrobiology laboratories, metagenomics is essen-

ial for understanding the effects of antibioticreatment of the human microbiome as a whole.urthermore, next-generation sequence analysisan facilitate testing of host susceptibility towardnfection and colonization if the high cost of thepparatus is considered to be a subsidiary issue forhe circumstances of each individual case. This cur-ent basic research development will become moreommon in diagnostic laboratories in coming years14].

icrobial volatile organic compoundetection

icrobial volatile organic compounds (MVOCs) areefined as a variety of compounds formed duringetabolism in bacteria and fungi. To date, more

han 200 different MVOCs have been identified,nd the diagnostic relevance of MVOCs has beenescribed in the literature. However, difficulties inhe reproducibility of diagnostic assays and the con-entrations of MVOCs cannot be overlooked [15].he most widely used MVOC detection technique ishe micro-weighing technique using vibration meth-ds, gas chromatography and metal chip electricalonductivity. This technology has been developed

ased on the design of a system that allows theinetic measurement of MVOC production by grow-ng bacteria; indeed, several bacterial species aredentifiable on the basis of their MVOC production

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profile. For instance, a study was conducted inwhich the diagnostic specificities varied from 100%for C. difficile to 67% for Enterobacter cloacae [16].This finding suggests that the diagnosis capability ofthis technique can be developed in the future foruse by clinical microbiology laboratories.

Raman spectroscopic analysis

The most evident contribution of spectroscopy toclinical microbiology has been in microbial epi-demiology. In recent years, Raman spectroscopicanalysis (RSA) has been validated for the bacterialtyping of different species. Although RSA has beenproposed to be capable of distinguishing virtuallyall bacterial species, definitive data are missing.RSA is a label-free, optical technology based on thenon-elastic scattering of light by the molecules. Thechange in wavelength is molecule-specific and canbe displayed in a Raman spectrum. These changescan be seen as spectroscopic fingerprints and canbe used to visualize the comprehensive molecularcomposition. Several studies concluded decisivelythat the typing of different strains belonging toa single species can be performed appropriately.The molecular typing of various species such asE. coli, Pseudomonas aeruginosa, Staphylococcus,and Klebsiella pneumoniae produced similar resultsthat were equal in quality to other gold standardtechniques [17].

Nanoparticles

A variety of methods have been introduced intoclinical microbiology over the course of the lastdecade. These methods clearly meet some diag-nostic standards and development is continuing.Moreover, many new technologies are making theirway into diagnostic microbiology, such as cell-basedbiosensors in the area of ‘nanodiagnostics’. Thistechnology uses cells as biochemical detectors withhigh sensitivity and specificity. Furthermore, therehas been an emergence of ‘theranostics’, whichappropriately combines attributes of diagnosticsand therapy to fight against infection. Recently, sil-ver nanoparticles (a noble metal known to exhibitstrong toxic effects against microbes and less tox-icity to humans) have been tested. A recent study[18] using electron microscopy revealed ‘pits’ in thecell walls of E. coli and other microbes that werecaused by the accumulation of silver nanoparticles.These ‘pits’ resulted in the alteration of membrane

morphology and increased membrane permeability,leading to cell death. Thus, this study clearly exem-plifies the role of nanoparticles in circumventingthe forthcoming threat of bacterial infections.

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Although there are numerous benefits of molec-lar techniques, the advantages of many conven-ional methods should not be overlooked. Currently,

well-equipped microbiology laboratory shouldave both reliable conventional methods and newolecular techniques, and staff should focus on

ssues such as cost, performance, durability andaintenance.

aboratory facilities in developingountries

he quality of the laboratory service dependspon various fundamental strategies, includingdequately trained laboratory staff, managementf the physical laboratory infrastructure, good lab-ratory practices, strict quality control, qualityssurance programs, and investment into new andmproved technology [19]. However, in many devel-ping countries there is scarcity of basic policiesequired for a quality functional laboratory. Labo-atory settings without proper infrastructure posehe greatest challenge for adequate performance.

lack of clean water and inadequate supply of elec-ricity and cold storage immensely limit the qualityf laboratory procedures [20].

aboratory management

he management of a high-quality clinical micro-iology laboratory is crucial for the control ofospital and community acquired infections. Dueo the lack of expertise in managing clinical micro-iology laboratories in developing countries, therehould be international guidance or training forhe staff; if possible, training programs should beonducted by high-quality laboratories from devel-ped countries. Careful review of the availableesources, foundation and facilities would expeditehe available support required. Furthermore, vis-ting various established laboratories that performdentical work can provide important information20]. Evaluation and accreditation of the labora-ory is also important; however, this process isependent on the overall cost and available fund-ng. Laboratory administration should take care ofheir internal staff and external communication sys-ems. The staff should receive medical evaluationn case of emergency, and medical records main-

enance should be performed appropriately [7].ffective communication between the laboratorynd health care providers should be supported,specially with the physician. Because the shared

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nformation is both qualitative and analytical, ade-uate provision for bidirectional interactions shoulde maintained. Telephone, email, facsimile andentral laboratory information systems are usefulor documentation [21].

aboratory networking systems

etworking between laboratories may increasenfection surveillance within a huge geographi-al region. The WHO supports the establishmentf national networks for the regular exchange ofnformation and proper support for the labora-ory. However, only a few countries have nationalnd international laboratory networks [22]. Thestablishment of an integrated laboratory systems very important to combat many infectious dis-ases. However, integrated systems are financiallyeglected in developing countries. Strengtheningaboratory systems in developing countries requiresffective strategic plans involving (a) legal and pol-cy framework, (b) institutional and managementramework, (c) human resources, (d) laboratoryuality management, (e) evaluation and moni-oring, (f) maintenance of equipment, and (g)aboratory structure and design [23].

aboratory biosafety and riskssessment

linical microbiology laboratories expose their staffnd the public to various clinical samples, whichay include various biohazards such as infectiousiseases. Therefore, it is important to develop aiosafety management program or operational planor the laboratory with regular training schemes,hereby ensuring the use of standard practices androcedures. Laboratory facilities are designed toeet certain biosafety levels that have designated

onstructional designs, containment accessibili-ies, equipment, operations and procedures thatre employed for risky biological agents. Sampleollection, transportation, handling, labeling anddentification should be performed according to

HO recommendations [24]. A laboratory biosafetylan should be maintained to reduce any accidentalelease of biohazardous material. The compre-ensive plan should include working guidelines,rimary care designs such as protective equipment

nd biosafety cabinets, and secondary care designsncluding laboratory facilities. Furthermore, theiosafety plan should promote the prevention ofaboratory-associated infections and environmental

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rotection. A biosafety committee should be estab-ished to gauge the success of the biosafety plan bymplementing and enforcing laboratory protocolsnd encouraging strict adherence to safety guide-ines. Because the identity of infectious agentsemains unknown during the initiation of microbialnvestigations, biosafety level-2 is recommendedor clinical microbiology laboratories [25,26].

aboratory accreditation

edical laboratories are regulated by governmentodies and strengthened by the standards validatedy other professional organizations. Accreditations an assessment process performed by an autho-ized body to ensure quality control and thessurance of the laboratories. This process pro-ides formal recognition that the laboratory canerform quality experiments. Laboratories test-ng human samples for the diagnosis, preventionr treatment of diseases are within the scopef the assessment [27]. All of the accredita-ion regulations are developed through consensus,ollaboration and consultation of health carerofessionals and health care authorities. Theaboratory standards are monitored by on-sitenspection, management requirements for patientesting, and follow-up actions. Although there areifferences in accreditation service proceduresnd programs, the overall assessment remainshe same [28]. The international organizationor standardization (ISO) is a global organiza-ion for national standards that is comprised of40 members across the globe. The Internationalccreditation Forum (IAF) and the Internationalaboratory Accreditation Cooperation (ILAC) dealpecifically with laboratories. The ILAC commis-ions tasks local bodies such as the Asia Pacificaboratory Accreditation Cooperation (APLAC) insia using ISO standards [29]. The standard guide-

ines for regulating quality laboratory managementre included in ISO 15189:2007, which can bebtained from the ISO website (http://www.so.org/iso/catalogue detail?csnumber=42641).

aboratory design

he establishment of a clinical microbiology lab-ratory in low-resource countries requires the

ollowing strategies: (a) requirement for physicalnfrastructure, (b) various assays, (c) supplemen-ation of reagents, (d) provision of equipmentnd maintenance, and (e) dedicated working

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stations. In developing countries, these key pointsare usually overlooked due to other issues. Themain requirement for designing a microbiologylaboratory is a clean and well-controlled environ-ment [30]. Surfaces contaminated with dust andaerosols and humid areas will be hazardous forthe microbiological procedures. Mechanical ven-tilation systems providing inward flow of air arerecommended to provide containment zones withincreased negative pressure. The reliable supplyand maintenance of gas and a standby electricitygenerator are preferred. Special attention shouldbe paid to the huge volume of microbes, overcrowd-ing equipment, infestation and unauthorized entry.Other laboratory features must also be addressed,such as dead space areas with no airflow and lowceilings that prevent the exclusion of contamina-tion during microbiology procedures [26]. Wastepipes, drainage, and ventilation systems manag-ing contaminated material should be maintainedefficiently. Even building material, such as a leak-ing roof that causes moisture, could be sources ofcontamination. The location of heavy equipmentshould facilitate the cleaning of the laboratorywalls and floors or may be placed on mobilebenches [31]. Illumination should be appropriatefor the safe conduct of laboratory work. Storageshould be appropriate for all supplies, reagents,hazardous chemicals and radioactive materials. Aholding zone for laboratory garments such as apronsand lab coats should be placed outside or possiblyat the entrance of the laboratory. Hand hygienebasins should be provided at the entry and exitpoints with alcohol rubs. Eating and drinking shouldnot be permitted within the laboratory becausethese activities may serve as the main sourceof contamination. If possible, every room in thelaboratory should have hand glove stations andmedical waste collection bags. Doors should havevision panels, be self-closing and have fire rat-ings [32]. Although there is no special guidelinefor the manner in which the space in a labo-ratory is used, an area of 350 ft/person withinthe laboratory would enable worker safety [26].The high risk of specimen—specimen contact whenperforming PCR-based assays requires specializedset-up. Four different physically separated work-ing areas are recommended, including an area forreagent preparation, PCR master mix preparation,nucleic acid processing and an amplification area.All of these working spaces should be kept free ofall other patient specimens [2]. Post-amplification

and ultraviolet (UV) photography should be per-formed in specialized rooms with a UV containmentarea. The use of UV aprons should be mademandatory within this area. The equipment used

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aboratory equipment

n resource-limited settings, there is lack ofecentralized molecular diagnostic testing andparse access to centralized medical facilities.ecause many molecular technique apparatusesre expensive to install in average microbiol-gy laboratories, cost-effective equipment can besed. Portable molecular technologies especiallyesigned for developing countries are available;or example, portable nucleic acid thermocy-lers are battery powered and cheaper thanench top units. These portable thermocyclersan perform PCR, reverse transcriptase-PCR andany formats of isothermal amplification, includ-

ng nucleic acid sequencing-based amplification,trand displacement amplification, rolling circlemplification, loop-mediated isothermal amplifica-ion, and helicase-dependent amplification. Therere valuable applications of these portable ther-ocyclers in diagnostic microbiology and biothreatetection. Various infectious agents have beendentified using these portable apparatuses, such asPV, N. gonorrhoeae, C. trachomatis, Treponemaallidum and T. vaginalis [33]. Several varieties ofortable apparatuses are commercially available;mportant evaluation criteria for each technologynclude maturity, power requirements, cost, sen-itivity, speed, and manufacturability. Ultimately,he needs of a particular market will lead to userequirements that drive the decision between avail-ble technologies.

There is a serious issue with instrument mainte-ance in developing countries because the lack ofunctional equipment will decrease the efficacy anduality of the laboratory procedures [22]. For qual-ty assurance, the facility should have documentedrocedures for the maintenance, calibration, ver-fication and validation of all equipment. Variousuccessful infection control programs have come to

halt in a number of developing countries due tohe lack of maintenance and service of the labora-ory equipment. Equipment failure and breakdownelays laboratory output; thus, equipment main-enance should be given priority. Selection ofquivalent brands is important and depends on theervice provided by the local dealer [19]. Some-

imes it is wiser to buy simple instrumentationhat is user friendly and less expensive ratherhan opting for automated instruments. Sophisti-ated electric equipment must also be avoided

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n developing countries unless it is essential dueo the irregular supply of electricity and the highost.

Furthermore, the laboratory staff should receiven-site training for equipment by the manufacturer34]. Thermocyclers are important instruments forolecular microbiology laboratories; any change

n the performance of thermocyclers will directlyffect the precision of the tests. Determinationnd documentation of cycling time or any errorshould be recorded; fluctuations in the cyclingimes should serve as warnings and be adjusted.dditionally, chiller, heater and block tempera-ure adjustments should be performed according tohe manufacturer’s recommendations [35]. Pipettesnd micro-pipettes are used in almost every stepf routine procedures; therefore, special atten-ion should be paid to their usage because theyre the prime source of errors. Pipette calibrationshould be performed regularly using recommendeduidelines. Special care should be given to theFGE apparatus, with proper cleaning of theank performed before and after every run andhe electrodes changed regularly. Most molecu-ar instruments are expensive. Recently, a reagentental agreement program has been introduced as aeans to acquire molecular instruments; however,

he availability of this program depends upon theanufacturer [36].

aboratory practice

eneral laboratory practice requires careful atten-ion, including regular cleaning and strict monitor-ng of all methods performed. The practice codes a list of the most essential procedures usedn basic good microbiology practices. It is impor-ant to develop written laboratory practices andethods for safe laboratory operations. Interna-

ional biohazard signs must be displayed on theaboratory door if the laboratory is working withsolates from risk group 2 or above. Only authorizedersonnel should be given access, and laboratoryoors should always be closed. All of the surfacereas should be cleaned before and after usage.orking stations, tables, refrigerators and reagent

ontainers should be regularly swabbed with 70%thyl alcohol [37]. The use of disinfectants recom-ended by the environmental protection agency

hould be promoted. Cleaning should be performed

n all surfaces, benches, racks, tiles, rockers andherever a spill occurs. For molecular procedures

especially PCR and PFGE), each working areahould contain dedicated reagents, materials and

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ping countries 523

ipetting devices. Reagents should be prepared inmall aliquots to avoid contamination [3].

aboratory personnel requirement

ncreasing the number of well-trained and skilledersonnel is a major limitation in developingountries. Although there is a steady demand forppropriate and adequate workers, governmen-al policies restrict the flow of skills. Laboratoryersonnel should be trained with all safety meas-res. Updating staff about the control measuresor laboratory biohazards is a key to preventingaboratory-acquired infections. An effective labo-atory program should be introduced by integratingafe laboratory practices into the basic trainingf the personnel [38]. Training should include allafety measures for handling hazardous materials,uch as aerosol inhalation, ingestion of specimens,ercutaneous exposure to sharp instruments, andandling and disposal of hazardous material. Thekills required for molecular techniques are morerecise and include micro-pipetting, DNA handlingnd disposal, software manipulation during analysisf the results and reporting [26]. Personal protec-ive equipment should always be used as a first linef safety:

) Protective laboratory coats must be worn at alltimes inside the laboratory.

) Hand protection should be followed by wearingdisposable gloves made of nitrile or chloroprene.

) Eye and face protection should be made com-pulsory whenever a splash or spill occurs.

aste management

n most low-income regions and especially in Asianountries the management of medical waste is aevere problem; only a handful of centers canrocess medical waste efficiently. Waste gener-ted by microbiology laboratories in the form ofpecimen samples, culture plates and tubes allarry a high potential risk of infection and needo be managed effectively. Most of the biomed-cal waste produced should be decontaminated,iscarded or incinerated [39]. Non-infectious mate-ials such as glassware are recyclable and can beterilized. Decontamination should be performed

y steam autoclaving. Contaminated sharp wastes,uch as hypodermic needles, disposable knives, andyringes, should be discarded in a puncture-proofag [32].

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Prospective instructions

Due to the lack of financial resources in develop-ing countries, partnerships between public, privateand commercial sectors should be maintained inorder to build a well-equipped laboratory. Fund-ing bodies should address the financial inequity,and an appropriate balance should be encouragedthat emphasizes laboratory infrastructure and man-agement. There should be a focus on accurateidentification and the use of highly reproduciblemethods. The funding bodies should target long-term training and education of future laboratorypersonnel. There should be improved communica-tion between the laboratory and clinicians, whichcould affect the accuracy of disease treatment.Policy makers and health care personnel mustunderstand that accurate and rapid identificationare important for the prevention and treatment ofdisease. It is very important to maintain a well-maintained clinical microbiology laboratory usingcurrent knowledge to control infections. Build-ing a molecular microbiology laboratory facility toprovide rapid, accurate and reliable tests will helphealth care staff to deliver more effective treat-ment regimens, thereby reducing mortality, overallexpenditure of health care resources and improv-ing the quality of health care services in developingcountries.

Funding

No funding sources.

Competing interests

None declared.

Ethical approval

Not required.

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