High rate of antimicrobial resistance and multiple mutations in the dihydrofolate reductase gene among Streptococcus pneumoniae isolated from HIV-infected adults in a community setting in Tanzania

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High rate of antimicrobial resistance and multiple mutations in the dihydrofolate reductase gene among Streptococcus pneumoniae isolated from HIV-infected adults in a community setting in Tanzania

Joel Manyahi

^a,b,c,

*, Sabrina Moyo

^a,c

, Said Aboud

^c

, Nina Langeland

^a,b

, Bjørn Blomberg

^a,b

aDepartmentofClinicalScience,UniversityofBergen,Bergen,Norway

bNationalCentreforTropicalInfectiousDiseases,DepartmentofMedicine,HaukelandUniversityHospital,Bergen,Norway

cDepartmentofMicrobiologyandImmunology,MuhimbiliUniversityofHealthandAlliedSciences(MUHAS),DaresSalaam,Tanzania

ARTICLE INFO

Articlehistory:

Received21April2020

Receivedinrevisedform3June2020 Accepted28June2020

Availableonline9July2020

Keywords:

Antimicrobialresistance Streptococcuspneumoniae HIV

Tanzania

Dihydrofolatereductase

ABSTRACT

Objectives: The aim of this study was to characterize molecular mechanisms of resistance to trimethoprimandotherantibioticsinStreptococcuspneumoniaeisolatesfromHIV-infectedadultsin DaresSalaam,Tanzania.

Methods: A total of 1877 nasopharyngeal swabs were collected and screened for pneumococcal colonizationfrom537newlydiagnosedindividualswithHIVatfourclinicvisitsduringa1-yearfollow- up from 2017–2018 as part of the randomized clinical trial CoTrimResist (ClinicalTrials.gov ID:

NCT03087890).

Results:Atotalof76pneumococcalisolateswereobtained.Ofthe70isolatesthatcouldbeserotyped,42 (60.0%)werevaccineserotypesincludedinpneumococcalconjugatevaccine23(PCV23).Themajorityof isolates (73.7%; 56/76) were non-susceptible to penicillin (MICs of 0.06–2m^g/mL). ^Isolates ^were frequently resistant to co-trimoxazole (trimethoprim/sulfamethoxazole) (71.1%) but less so to azithromycin(22.4%),erythromycin(21.1%),chloramphenicol(18.4%),tetracycline(14.5%),clindamycin (10.5%)and levoﬂoxacin(0%). Moreover,26.3%weremultidrug-resistant (resistantto3 antibiotic classes).Vaccine-typepneumococciwereresistanttomoreclassesofantibiotics,weremorefrequently resistanttoerythromycin,azithromycin,clindamycinandtetracycline,andhadhigherMICstopenicillin (median,0.19m^g/mL;^range,^0.002–1.5m^g/mL)^compared^withnon-vaccineserotypes(median,0.125m^g/

mL;range,0.012–0.25m^g/mL)^(P⁼^0.003).Co-trimoxazole-resistantisolatescarriedfrom1to11different mutationsinthedihydrofolatereductase(DHFR)gene,mostcommonlyIle100Leu(100%),Glu20Asp (91.8%),Glu94Asp(61.2%),Leu135Phe(57.1%),His26Tyr(53.1%),Asp92Ala(53.1%)andHis120Gln(53.1%).

Conclusion: Streptococcus pneumoniae isolated from HIV-diagnosed patients were frequently non- susceptibletopenicillinandco-trimoxazole.MostisolatescarriedmultiplemutationsinDHFR.

org/licenses/by-nc-nd/4.0/).

1.Introduction

Streptococcuspneumoniaeisacommoncauseofinvasiveand non-invasive diseases. Unfortunately, pneumococcal disease remainsaprimarycauseofmorbidityandmortalityinimmuno- competentand immunodeﬁcientpopulations[1,2].Nasopharyn- gealcolonizationwithS.pneumoniaeisconsideredaprerequisite bothforinvasiveandnon-invasivepneumococcaldiseases[3,4].In humanimmunodeﬁciencyvirus(HIV)infection,widespreaduseof

co-trimoxazole(trimethoprim/sulfamethoxazole)andotheranti- bioticshasbeenassociatedwithincreasedcarriageofmultidrug- resistant (MDR) bacteria, including MDR S. pneumoniae [5–8].

Infectionswithpenicillin-resistantstrainsaredifﬁculttotreatand areassociatedwithincreasedmorbidityandmortalityaswellas increasedhealthcarecosts[2].

FewstudieshavebeencarriedoutinTanzaniaonpneumococcal nasopharyngealcarriage.Amongthese,somehavefoundthatHIV- exposedandnon-exposedchildrenhavehighratesofS.pneumoniae resistanttocommonlyprescribedantibiotics,includingco- trimoxazole and penicillin [9–11]. Likewise, nasopharyngeal S.

pneumoniae isolates with non-susceptibility to commonly pre- scribedantibioticssuchaspenicillin,macrolidesandtetracycline havebeenreportedpreviouslyinhealthychildreninDemocratic

* Correspondingauthorat:MuhimbiliUniversityofHealthandAlliedSciences,P.

O.Box65001,DaresSalaam,Tanzania.

E-mailaddresses:[email protected],[email protected](J.Manyahi).

http://dx.doi.org/10.1016/j.jgar.2020.06.026

2213-7165/©2020TheAuthors.PublishedbyElsevierLtdonbehalfofInternationalSocietyforAntimicrobialChemotherapy.ThisisanopenaccessarticleundertheCCBY-NC- NDlicense(http://creativecommons.org/licenses/by-nc-nd/4.0/).

ContentslistsavailableatScienceDirect

Journal of Global Antimicrobial Resistance

j o u r n a lh o m e p ag e :w w w . e l s e vi e r . c o m / l o c a t e/ j g a r

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Republicof Congo[12] and HIV-infectedpatientsin Cameroon [13].

Inrecentyears,trimethoprim hasrarelybeenusedalonein the treatment of bacterial infections, with the exception of urinarytract infections.The combination of trimethoprim and sulfamethoxazole (co-trimoxazole) has been used extensively insteadinthetreatment ofrespiratorytractinfections,urinary tractinfectionsandgastrointestinaltractinfections [14].Resis- tance to either trimethoprim or sulfamethoxazole renders bacteria resistant to co-trimoxazole as well. Resistance to trimethoprimand sulfamethoxazoleis conferredbyacquisition of mutations in the dihydrofolate reductase (DHFR) and dihydropteroate synthase (DHPS) genes, respectively. Studies haveshownthatinS.pneumoniae,asinglesubstitutionofamino acidisoleucineatposition100withleucineinDHFRissufﬁcient toconferresistancetotrimethoprim[15,16].Arecentreportfrom Malawifoundthat substitutionof amino acidatposition92 of DHFRwithoutIle100Leucouldalsoconferresistancetotrimeth- oprim[17].However,inpreviousstudiesmultiplemutationshave beenobservedintheDHFRgeneinS.pneumoniae,althoughtheir roleinconferringtrimethoprimresistanceisnotwellknown.

InTanzania,nopreviousstudyhasassessedthemolecularbasis ofco-trimoxazoleresistanceinS.pneumoniaeisolatedfromHIV- infectedadults.Moreover,therearelimiteddataonS.pneumoniae colonization and antimicrobial resistance among HIV-infected adultsfromcommunitysettingsinTanzania.Theaimofthisstudy wastodeterminethemolecularmechanismsconferringtrimeth- oprimresistancein S.pneumoniaeaswell astounderstandthe antimicrobialresistancepatternsofS.pneumoniaecolonizingthe nasopharynxofHIV-infectedadultsfromacommunitysettingin Tanzania.

2.Materialsandmethods

2.1.Studyparticipants

NewlydiagnosedadultswithHIVwererecruitedfromsixHIV care and treatment clinics at Amana, Mwananyamala, Temeke RegionalReferral,PASADA,MbagalaandMnaziMmojahospitalsin DaresSalaam(Tanzania)aspartoftherandomizedclinicaltrial CoTrimResist(ClinicalTrials.gov ID:NCT03087890)toassess any effect of prolonged co-trimoxazole prophylaxis on emerging antimicrobialresistanceinHIV patients(datanotyetanalyzed).

Atotalof537participantswererecruitedatbaselinebetweenApril 2017andMay2018andwerefollowed-upfor1year.

2.2.Microbiologicalmethods

2.2.1.Specimencollection,isolationandidentiﬁcationof Streptococcuspneumoniae

A total of 1877 nasopharyngeal swabs were collected at baseline, at Day 14 and at Weeks 24 and 48. Nasopharyngeal swabswerecollectedbyatrainedclinicianfromeachhealthcare facilityusingSigmaTranswab¹andweretransportedimmediately in liquid Amies transport medium [Sigma Transwab¹ PF with LiquidAmies;MWECo(Bath)Ltd.,Corsham,UK]inacoolboxat 4C.Uponreceiptinthelaboratory,nasopharyngealsampleswere immediately inoculated onto 5% sheep blood agar and were incubated at 37C in 5% CO2 for 24h. Identiﬁcation of S.

pneumoniaewas made by colonial morphology, presence of α- haemolysis,optochinsusceptibilityandbilesaltsolubility.

2.2.2.Antimicrobialsusceptibilitytesting

AntimicrobialsusceptibilitytestingwasperformedonMueller– Hintonagarsupplementedwith5%sheepbloodandplateswere incubatedat 35C in 5% CO2 for 20–24h. Minimum inhibitory

concentrations(MICs)forpenicillin,azithromycin andtrimetho- prim/sulfamethoxazole were determined by Etest (bioMérieux, Marcy-I’Étoile, France). Antimicrobial susceptibility testing for chloramphenicol, tetracycline, erythromycin, clindamycin and levoﬂoxacin was performed by the Kirby–Bauer disk diffusion method.Antimicrobialsusceptibilitytestresultswereinterpreted according to Clinical and Laboratory Standards Institute (CLSI) guidelines.MDRwasdeﬁnedasbacteriaresistanttothreeormore classes/categoriesofantibiotics[18].

2.2.3.Nucleicacidextraction

Frompuregrowth,5–10coloniesweresuspendedin500

m

^L

of phosphate-bufferedsaline(PBS).DNAwasextractedusing a MagNA Pure LC instrument (Roche Diagnostics, Mannheim, Germany) using a Total Nucleic Acid Isolation Kit (Roche Diagnostics). Extracted DNA was eluted in 100

m

^L ^of ^elution

buffer. DNA templates were stored at 20C until further analysis.

2.2.4.PCR

PCR for detection of the DHFR gene was performed using 2QuantiTect¹MultiplexPCRNoROXMasterMix(QIAGEN)and amplificationwas carriedout on a GeneAmp^TM 9700Thermo- cycler(AppliedBiosystems,FosterCity,CA,USA).Thefollowing setofprimerswasusedaspreviouslydescribed[16]:5⁰-TGTAAG CTATTCCAAACCAG-3⁰and5⁰-CTACGTTCCATTAGACTTCC-3⁰ (PCR product, 760bp). PCR conditions consisted of initial denaturation at 95C for 15min, followed by denaturation at 94Cfor60s,annealingat45Cfor 30sandextensionat72C for1min,andafinalextensionat72Cfor7min.Afinalreaction volume of 25

m

^L ^consisted ^of ^the ^following: ^12.5

m

^L ^of ² QuantiTect¹ Multiplex PCR NoROX Master Mix, 1

m

^L ^of ^each

primer (0.4

m

^M), ^8.5

m

^L ^of ^RNase-free ^water ^and²

m

^L ^of ^DNA

template. Ampliﬁed PCR products were analyzed by gel electrophoresis.

2.2.5.DNAsequencing

AmpliﬁedPCRproductswerepuriﬁedandbothstrandswere sequencedusing thesame primersas forPCR. Sequencingwas performed using an ABI PRISM 3730 DNA Analyzer (Applied Biosystems)withaBigDye^TMTerminatorv.1.1CycleSequencingKit (Applied Biosystems).SnapGene¹ v.5.0.7 software(GSL Biotech LLC,Chicago,IL,USA)wasusedtoassemble,editandanalyzethe sequences.

2.2.6.SerotypingofStreptococcuspneumoniaeisolates

Serotyping was performed from an overnight growth of S.

pneumoniaeon5% sheepblood agarusingacommercialkitfor latexagglutination(Immulex^TMPneumotestKit;SSIDiagnostica A/S, Hillerød, Denmark). The agglutination kit contains latex particles coated withrabbit antibodiesthat reactwith speciﬁc pneumococcalcapsularpolysaccharides.Performanceand inter- pretation of the test results followed the manufacturer's instructions.

2.3.Statisticalanalysis

Categorical variableswerepresentedas theproportion,and continuous variables were presented using the median and range. Proportions of resistant bacteria between vaccine- and non-vaccineserotypeisolateswerecomparedbyχ²test,andthe medians of MICs were compared by Wilcoxon rank-sum test usingSTATAv.16.0(StataCorpLLC,CollegeStation,TX,USA).AP- value of <0.05 was deﬁned as the cut-off for statistical signiﬁcance.

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3.Results

3.1.Streptococcuspneumoniaeisolates

A total of 76 S. pneumoniae were isolated from 1887 nasopharyngeal swabs. The number of S. pneumoniae isolates obtainedatdifferenttimepointswas asfollows:20atbaseline (n=537 swabs); 13 at Day 14 (n=509 swabs); 17 at Week 24 (n=436swabs);and26atWeek48(n=395swabs).

3.2.SerotypingofStreptococcuspneumoniaeisolates

Themajorityofisolates(55.3%,42/76)wereserotypespresent inthepneumococcalconjugatevaccine23(PCV23),whilst36.8%

(28/76)werenon-vaccineserotypesand7.9%(6/76)couldnotbe typedbythemethodused.Themostfrequentconjugatevaccine serotypeswere19(9/42),3(8/42),7(6/42)and15(4/42)(Fig.1).

3.3.Antimicrobialsusceptibilitytesting

Table1 shows thenumberand percentage ofS.pneumoniae resistanttodifferentantibiotics.Themajorityofisolates(73.7%;

56/76) werepenicillin-non-susceptible (MICs of 0.06–2

m

^g/mL),

butnoisolatewasfullypenicillin-resistant.Mostisolateswerealso resistant to co-trimoxazole (71.1%; 54/76). In addition, co- trimoxazoleresistance wassigniﬁcantlymorefrequentinpneu- mococciwithnon-susceptibilitytopenicillin(82.1%;46/56)thanin fullypenicillin-susceptibleisolates(40.0%;8/20)(P<0.001).

Rates of resistanceto azithromycin, erythromycin, chloramphenicol,tetracyclineandclindamycinwere22.4%(17/76),21.1%

(16/76),18.4%(14/76),14.5%(11/76)and10.5%(8/76),respectively.

Allisolatesweresusceptibletolevoﬂoxacin.Approximatelyone- quarteroftheisolates(26.3%;20/76)wereMDR.Vaccine-typeS.

pneumoniaewereresistanttomoreclassesofantibioticscompared withnon-vaccineserotypeS.pneumoniae[medianof3(range3–5) vs.2.5(range0–5);P=0.03]andweremorefrequentlyresistantto erythromycin(33.3%vs.7.1%; P=0.011),azithromycin (33.3%vs.

10.7%; P=0.031), clindamycin (19.0% vs. 0.0%; P=0.014) and tetracycline(23.8%vs.0.0%;P=0.005).Althoughvaccineserotype isolatesdisplayedsigniﬁcantlyhigherMICstopenicillin(median, 0.19

m

^g/mL;^range,^0.002–1.5

m

^g/mL)^compared^withnon-vaccine serotypeisolates(median,0.125

m

^g/mL;^range,^0.012–0.25

m

^g/mL)

(P=0.003),therewerenosigniﬁcantdifferencesintheproportions of isolates with non-susceptibility to penicillin. Neither co- trimoxazoleMICsnortheproportionofco-trimoxazoleresistance

was signiﬁcantly different between vaccine and non-vaccine serotypes.

3.4.Mutationsindihydrofolatereductase(DHFR)

Among the61 S. pneumoniaeisolates witha co-trimoxazole MIC>2

m

^g/mL,⁴⁹^weresuccessfullysequenced andhadnucleo- tidesequencesavailableforanalysis.

Co-trimoxazole-resistantisolatescarriedfrom1to11different mutations in the DHFR gene, with the majority(71.4%; 35/49) having5to9mutations(Table2).Themostcommonmutations conferring resistance to trimethoprim were substitution of Ile100Leu(100%),Glu20Asp(91.8%),Glu94Asp(61.2%),Leu135Phe (57.1%),His26Tyr(53.1%),Asp92Ala(53.1%)andHis120Gln(53.1%) (Table3).Therewasnodifferenceinthenumberofmutationsin the DHFR gene between vaccine and non-vaccine serotype pneumococci (median,5.5;range, 0–11 forboth;P=0.4).There wasnosigniﬁcantassociationbetweenco-trimoxazoleMICsand thenumberortypesofmutationsobserved.

4.Discussion

ThisstudydemonstratedthatS.pneumoniaeisolatedfromthe nasopharynxofHIV-infectedadultswerefrequentlyresistantto commonly prescribed antibiotics in resource-limited settings.

Approximatelyone-quarteroftheisolateswereMDRbacteria.The non-susceptibilityofS. pneumoniaetopenicillinand co-trimox- azoleisworrisomeastheseantibioticsarecommonlyusedasﬁrst- line treatment for pneumococcal pneumonia in resource-con- strainedcountries.

The high rate of co-trimoxazole-resistant S. pneumoniae colonizingthenasopharynxobservedinthisstudyisinlinewith ﬁndingsfromHIV-infectedpopulationsinTanzania[9]andother resource-limited settings [19,20]. Co-trimoxazole is widely available over the counter in resource-constrained counties.

Irrational use of co-trimoxazole could possibly explain the observed ﬁnding. Previous studies have indicated that co- trimoxazoleuseincreasestheriskofcarriageofco-trimoxazole- resistantS.pneumoniae[21–23].

Previous studies have found that trimethoprim resistance mutations,morethansulfamethoxazoleresistancemutations,are correlatedwithresistancetotrimethoprim/sulfamethoxazole(co- trimoxazole) [15]. In the current study, theDHFR genes of co- trimoxazole-resistant S. pneumoniae(n=49) weresequencedto determinealterationsinthechromosomalDHFRgeneconferring pneumococcalresistancetotrimethoprim.Overall,substitutions weredetectedinupto11aminoacidpositions;thesesubstitutions were far fewer than those reported previously among 68 trimethoprim-resistant S. pneumoniae in North America [16].

PreviousstudieshavedemonstratedthatsubstitutionofIle100Leu is critical for development of trimethoprim resistance in S.

pneumoniae[15,17,24].Inthecurrentstudy,itwasalsofoundthat allsequencedco-trimoxazole-resistantpneumococcalisolateshad thesamemutationofIle100Leu.Mutationsatotherlocationsare thoughttoincrease theMICof trimethoprim[17].Mutationsof Glu20Asp(91.8%),Glu94Asp(61.2%)andLeu135Phe(57.1%)were alsofrequentlyfoundasdocumentedpreviously[16,25].Arecent studyinMalawi[17]reportedthatmutationatresidue92without substitutionofIle100LeuwasassociatedwithanincreaseMICof trimethoprim-resistant pneumococci. In the current study, a number ofisolates withsubstitutionat residue 92werefound, butallofthemalsohadtheIle100Leumutation.Thisstudydidnot investigatethemechanismofresistancetosulfamethoxazole,the other ingredient of trimethoprim/sulfamethoxazole (co-trimoxazole). However, the study conﬁrmed the high prevalence of known resistance mutationsin the DHFRgene associated with Fig.1.Serotypesdistributionamong nasopharyngealStreptococcuspneumoniae

isolatesfromHIV-infectedadultsinTanzania(n=76).

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trimethoprim resistance in S. pneumoniae isolates. Hence, co- trimoxazolemightnotbeeffectivetotreatpneumococcalinfection inHIV-infectedindividuals.

The rate of penicillin-non-susceptibility in isolates of S.

pneumoniae(73.7%)inthecurrentstudyiscomparablewiththat foundamongchildreninthepre-PCVera(67.8–69.2%)[9,10]and post-PCVera(31–53%)[11]inTanzania.However,inthecurrent studyamuchhigherratewasfoundthaninarecentstudyinGhana that reported only 25.9% of penicillin-non-susceptible S.

pneumoniaefromthenasopharynxamongHIV-infectedindividu- alsinthePCVera[19].AlthoughnoneoftheS.pneumoniaeisolates hadahighlevelofresistancetopenicillin(>2

m

^g/mL),^the^current

ﬁndingquestionstheappropriatenessofusingpenicillinforthe treatmentofseverepneumococcalinfectionssuchasmeningitisin Tanzania.However,athighintravenousdoses,itcanstillbeusedto treat non-meningeal pneumococcal infections. Fully penicillin- resistantS.pneumoniae havebeenreportedelsewherein Africa [26,27].AlthoughtheyarecurrentlyuncommoninTanzania,there isneedforcontinuoussurveillancetomonitortheemergenceof fullypenicillin-resistantstrains.

Interestingly, vaccine serotype isolates of S. pneumoniae showedhigherratesofresistancetoerythromycin,azithromycin, clindamycinandtetracycline.Thebackgroundforthismaybethat vaccineserotypewereselectedforuseinvaccinesbecausethey werequitevirulent.Withahistoryofsuchvirulence,theancestor bacteriumofthevaccineserotypesmayhavecausedmuchillness andelicitedmoreantibioticuse,whichinturnmayhaveselected for re-emerging drug resistance. Both tetracycline andmacro- lides, particularlyerythromycin, have beenused extensively as theyareoralmedicineswithbroad-spectrumactivity[28].What weseenowmaythusbetheresultofantibioticuseinthepre- vaccine era. Ourobservation is similar to a previous study on clinical isolates which found that vaccine serotypes displayed moremultidrugresistancecomparedwithnon-vaccineserotypes [29].

Thecurrentﬁndingsareinlinewithotherstudiesfromdifferent populationsofchildreninTanzania[9–11]aswellasstudiesfrom GhanaandCameroonofnasopharyngealcarriageinHIV-infected adults[13,19]andchildren[26],whichhavereportedlowratesofS.

pneumoniaeresistanttoerythromycininthePCVera.

The relatively low rates of resistancetoazithromycin docu- mented in this study are also comparable with the ﬁndings reportedpreviouslyinsemi-urbansettingsinTanzania[30,31].Ina previous study conducted in Tanzania, mass administration of azithromycin was foundtocorrelatewith anincreasedrisk for nasopharyngealcarriageofazithromycin-resistantS.pneumoniae [32]. Based on the current ﬁndings, both azithromycin and erythromycincouldstillbeanoptionfornon-severeS.pneumoniae infections in HIV-infected patients. However, rational use of macrolidesneedstobeadvocatedinthecountry,asobservedina previous6-monthcohortstudyincentralTanzania[32].

5.Conclusions

Streptococcus pneumoniae isolated from HIV-infected adult patientswerefrequentlynon-susceptibletopenicillinandresis- tant toco-trimoxazole.Themajorityofthese isolatesdisplayed MDRtraits.MostisolatescarriedmultiplemutationsintheDHFR geneandallcarriedtheIle100Leusubstitution.

Table1

DistributionofStreptococcuspneumoniaeresistancetovariousantibioticsinHIV-infectedadultpatients.

Antibiotic %(n) P-value*

All(n=76) Vaccineserotype(n=42) Non-vaccineserotype(n=28) Non-typeable(n=6)

Penicillin-non-susceptible^a 73.7(56) 78.6(33) 64.3(18) 83.3(5) 0.188

Co-trimoxazole-resistant^a 71.1(54) 76.2(32) 67.9(19) 50.0(3) 0.442

Azithromycin-resistant^a 22.4(17) 33.3(14) 10.7(3) 0.0(0) 0.031

Erythromycin-resistant^b 21.1(16) 33.3(14) 7.1(2) 0.0(0) 0.011

Clindamycin-resistant^b 10.5(8) 19.0(8) 0.0(0) 0.0(0) 0.014

Tetracycline-resistant^b 14.5(11) 23.8(10) 0.0(0) 16.7(1) 0.005

Chloramphenicol-resistant^b 18.4(14) 21.4(9) 7.1(2) 50.0(3) 0.108

Levoﬂoxacin-resistant^b 0.0(0) 0(0) 0(0) 0(0) –

aDeterminedbyEtest.

b DeterminedbytheKirby–Bauerdiskdiffusionmethod.

*P-valuefordifferencebetweenvaccineandnon-vaccineserotypeisolates(χ²test).

Table2

Prevalence of mutations in and median co-trimoxazole minimum inhibitory concentrations(MICs)ofco-trimoxazole-resistantStreptococcuspneumoniaisolates (n=49)fromHIV-infectedadultpatientsinTanzania.

No.ofmutations n(%) MedianMIC(range)

1 2(4.1) 3(3–3)

2 2(4.1) 19(6–32)

3 2(4.1) 4(4–4)

4 2(4.1) 5(2–8)

5 5(10.2) 8(4–32)

6 9(18.4) 16(4–32)

7 8(16.3) 5(2–16)

8 9(18.4) 3(2–32)

9 4(8.2) 3.5(2–7)

10 2(4.1) 10(4–16)

11 4(8.2) 7(4–8)

Table3

Typesofmutationandmedianco-trimoxazoleminimuminhibitoryconcentrations (MICs)ofco-trimoxazole-resistantStreptococcuspneumoniaeisolates(n=49)from HIV-infectedadultpatientsinTanzania.

Mutation Prevalence [n(%)]

MedianMIC(range)(m^g/mL) ^P- value*

Mutationpresent Mutationabsent

E20D 45(91.8) 6(2–32) 3(3–32) 0.4

H26Y 26(53.1) 6(2–32) 4(2–32) 0.7

P70L 5(10.2) 4(3–32) 6(2–32) 0.5

P70S 17(34.7) 4(2–32) 6(2–32) 0.2

A78T 16(32.7) 4(2–16) 6(2–32) 0.06

Q81H 12(24.5) 4(2–32) 7(2–32) 0.1

Q81Y 11(22.4) 6(2–8) 5(2–32) 0.5

V83I 15(30.6) 4(2–32) 6(2–32) 0.7

Q91H 5(10.2) 8(4–16) 4(2–32) 0.3

D92A 26(53.1) 6(2–32) 4(2–32) 0.9

D92V 3(6.1) 4(4–32) 5(2–32) 0.7

D92G 13(26.5) 7(2–32) 4(2–32) 0.9

E94D 30(61.2) 6.5(2–32) 4(2–32) 0.2

I100L 49(100.0) 6(2–32) N/A –

H120Q 26(53.1) 6(2–32) 4(2–32) 0.7

L135F 28(57.1) 6.5(2–32) 4(2–32) 0.4

N/A,notapplicable.

*Wilcoxonrank-sumtest(Mann–Whitney).

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Funding

This study was supported by Helse Bergen HF, Haukeland UniversityHospital,Norwaythroughprojectnumber912132.The funders had no role in the study design, data collection and analysis,thedecisiontopublish,orpreparationofthemanuscript.

Competinginterests Nonedeclared.

Ethicalapproval

EthicalapprovaltoconductthestudyinTanzaniawasobtained fromtheMuhimbiliUniversityofHeathandAlliedSciencesSenate ResearchandPublicationsCommittee[Ref.No.2015-10-27/AEC/

Vol.X/54],theNationalEthicsHealthResearchEthicsCommittee [Ref.No.NIMRlHQ/R.SaJVol. 1X12144],theTanzaniaFoodandDrug Authority[Ref.No.TZ16CT007]and theRegional Committeefor MedicalandHealthResearchEthicsofWesternNorway[Ref.No.

REK2015/540].Writteninformedconsentwasobtainedfromeach studyparticipantpriortoenrolmentinthestudy.

Acknowledgments

The authors acknowledge members of the Department of ClinicalScience,UniversityofBergen(Bergen, Norway)fortheir technicalsupportduringthemolecularstudy.

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