Common Lab investigationsCommon Lab investigations in Pediatric Practice in Pediatric Practice

Dr.S.Srinivas. DCHDr.S.Srinivas. DCH

1.CBC – Few practical points1.CBC – Few practical points

Commonest lab test in clinical practiceCommonest lab test in clinical practice Results are not specific to a particular disease Requires interpretation based on patients clinical condition Peripheral smear -most important but negleted part of CBC All three cell lines and subtypes of WBC need proper All three cell lines and subtypes of WBC need proper

evaluationevaluation

1.1. WBC count and DCWBC count and DC

2.2. RBC count ,Hb% and IndicesRBC count ,Hb% and Indices

3.3. Platelet count and IndicesPlatelet count and Indices

LeucocytesLeucocytes

Neutrophils : 60% (>70%-Abnormal)

Lymphocytes : Upto 40%

WBC WBC Normal range varies with ageNormal range varies with age

Increased WBC Increased WBC

Acute stress of various causesAcute stress of various causes Infection, tissue necrosis Infection, tissue necrosis bone marrow malignancies, bone marrow malignancies,

inflammation inflammation

Decreased WBC Decreased WBC InfectionsInfections conditions or medications that conditions or medications that

suppress or weaken the immune suppress or weaken the immune system or bone marrowsystem or bone marrow

Neutrophils Neutrophils -60% -60% First line of defense against infection First line of defense against infection Two types: Two types: Bands (0-3%) - immature Bands (0-3%) - immature Segs (31% - 57%) – mature Segs (31% - 57%) – mature Neutrophilic leucocytosis with >20% band-Neutrophilic leucocytosis with >20% band- Acute bacterial infectionAcute bacterial infection –(Not a rule)–(Not a rule)Neutrophilia indicates stress of Neutrophilia indicates stress of Acute infection-Bacterial/viralAcute infection-Bacterial/viral InflammationInflammation Others – Burns ,acute asthmaOthers – Burns ,acute asthmaNeutropaenia indicates marrow suppressionNeutropaenia indicates marrow suppression Typhoid,Viral infectionsTyphoid,Viral infections

Absolute Neutrophil Count (ANC) Absolute Neutrophil Count (ANC)

The real number of white blood cells (WBCs) The real number of white blood cells (WBCs) that are neutrophils. that are neutrophils.

A normal ANC is about 3,000-5,000. A normal ANC is about 3,000-5,000. <1,000 greater risk for infection. <1,000 greater risk for infection.

Lymphocytes Lymphocytes (Upto 40%)(Upto 40%)

Lymphocytosis (>5000/mm)Lymphocytosis (>5000/mm) Viral – Infectious mononucleosis, mumps, rubellaViral – Infectious mononucleosis, mumps, rubella Chronic infections -TBChronic infections -TB ALLALL

Lymphocytopenia (<2700)Lymphocytopenia (<2700) ImmunodeficiencyImmunodeficiency

Eosinophils Eosinophils (1-4%)(1-4%) Eosinopenia <50/c.mmEosinopenia <50/c.mm Typhoid/Acute viral/bacterialTyphoid/Acute viral/bacterial Eosinophillia >500/c.mmEosinophillia >500/c.mm Parasitic infestationsParasitic infestations Basophilia >100/c.mmBasophilia >100/c.mmViral –Chickenpox,InfluenzaViral –Chickenpox,Influenza Monocytosis >800/c.mmMonocytosis >800/c.mmInfectious mononucleosisInfectious mononucleosisSubacute bacterial endocarditisSubacute bacterial endocarditisMalariaMalaria

Leukocyte abnormalities –limitations…

TC -low predictive value due to wide range of normal count (8000 to 20,000/cu.mm).

Non-specific-infective /non-infective inflammation Non specific-bacterial /viral. Leukamoid reaction (WBC count > 50,000) should

not be mistaken for malignancy like leukemia.

(Leukocyte alkaline phosphatase increased in LR)

RBC indicesRBC indices Hb% - Degree of anemiaHb% - Degree of anemia RBC countRBC count MCV – Cell size (75-95)MCV – Cell size (75-95) RDW (11.5%-14.5%) High =AnisocytosisRDW (11.5%-14.5%) High =Anisocytosis

MCV- Low MCV -Normal MCV-High

RDW-Normal RDW-High Chronic diseaseSpherocytosis

RDW-Normal RDW-High

Thal. Trait IDAThal .major

Aplasia B12-deficiencyFolate -deficiency

Platelets – Known facts…Platelets – Known facts…ThrombocytosisThrombocytosis Viral infectionsViral infections Inflammatory disordersInflammatory disorders IDAIDA Lab errorLab errorThrombocytopaeniaThrombocytopaenia Viral (including dengue)Viral (including dengue) TyphoidTyphoid MalariaMalaria ITP/Aplastic anemia/LeukemiaITP/Aplastic anemia/Leukemia DICDIC SepsisSepsis

MPV reflects the average size of plateletsMPV reflects the average size of platelets Predicts functionally good plateletsPredicts functionally good platelets High MPV in a person with a low platelet count High MPV in a person with a low platelet count

suggests the bone marrow is producing platelets suggests the bone marrow is producing platelets and releasing them into circulation rapidly. and releasing them into circulation rapidly.

Low MPV with low platelet counts - due to a Low MPV with low platelet counts - due to a disorder affecting production of platelet by the disorder affecting production of platelet by the bone marrow bone marrow

PDW reflects how uniform the platelets are in size. PDW reflects how uniform the platelets are in size. Normal PDW - platelets are mostly same sizeNormal PDW - platelets are mostly same size High PDW - platelet size varies in sizeHigh PDW - platelet size varies in size

Neglected part – MPV & RDW

HbHb WBCWBC PP LL EE PltPlt DiseaseDisease

NN ++++++ ++++++ 00 NN Acute bacterial infAcute bacterial inf

NN ++++++ ++++++ NN ++++ Sys.Inflammatory disSys.Inflammatory dis

N N ++++ ++++ 00 N/LN/L Acute viral infAcute viral inf

NN LL ++++ oo LL TyphoidTyphoid

NN +/-+/- ++++ NN N/LN/L Chronic infectionsChronic infections

LowLow +/-+/- ++++ NN N/LN/L MalariaMalaria

LowLow ++++++ ++++++

NN LowLow A L LA L L

HighHigh +/-+/- ++++ 00 LowLow DengueDengue

CBC in common clinical conditionsCBC in common clinical conditions

AlertsAlerts

High Hb +Pcv(>20%) –Dengue feverHigh Hb +Pcv(>20%) –Dengue fever Low WBC + High Hb - DengueLow WBC + High Hb - Dengue High WBC + High lymphocytes – ALLHigh WBC + High lymphocytes – ALL Low WBC + Low platelets + low Hb – Bone Low WBC + Low platelets + low Hb – Bone

marrow diseasemarrow disease Low lymphocytes <2700 -ImmunodeficiencyLow lymphocytes <2700 -Immunodeficiency

CBC in fever-limitationsCBC in fever-limitations

CBC done very early during a febrile illness does not CBC done very early during a febrile illness does not help much help much

Interpret only in conjunction with the clinical pictureInterpret only in conjunction with the clinical picture It should be repeated if fever persists without diagnosisIt should be repeated if fever persists without diagnosis Serial counts (eg) DengueSerial counts (eg) Dengue

Case 1Case 1 5 yr old boy –Acute onset of high grade fever-2days5 yr old boy –Acute onset of high grade fever-2days Interfebrile state –SickInterfebrile state –Sick Poor response to supportive treatmentPoor response to supportive treatment D2 –CBC-D2 –CBC-HbHb 11.5g% HCT-34 11.5g% HCT-34 TCTC-7600 -7600 DCDC- - PP62 62 LL36 36 EE 0 0 Plt-1,60,000 , Plt-1,60,000 , Urine R/E –Normal D3-C/O Abd.pain,headache,2 episodes of vomitingD3-C/O Abd.pain,headache,2 episodes of vomiting Sick looking,Temp – 104 F ,Moderate dehydration Sick looking,Temp – 104 F ,Moderate dehydration CRFT-3sec ,Low pulse volumeCRFT-3sec ,Low pulse volume Flushing-Flushing-white island in the red seawhite island in the red sea Admitted –Supportive treatment –Rpt CBC -Admitted –Supportive treatment –Rpt CBC -Hb-13.4g%

HCT 41.4 TC 3100 DC P38 L 61E0 Plt- 95,000

Provisional diagnosisProvisional diagnosis

?Dengue fever?Dengue fever Treatment - WHO guidelines/frequent assessment

Logical Lab investigations in Suspected dengue

1.For assessment and management Serial –Hb%, Hct, Platelet count Na,BUN,LFT DIC Panel X-ray chest, USG abdomen

2.For confirmation of diagnosis Serological tests

CBC in dengue Serial Hb%, Hct, Platelets – Atleast every 24 h Q 4 h in severe cases of dengue. Rise in Hct level > 20% from baseline is a sign of

haemoconcentration - Shock Rapid decrease in platelet count +rising Hct =

progress to the plasma leakage/critical phase Leukopenia(<5000)+Positive tourniquet test in

Dengue endemic area-Positive pred.value-70-80%

Confirmatory testsConfirmatory tests

Diagnosis is clinical …Diagnosis is clinical … Monitoring with simple testsMonitoring with simple tests

Confirmatory tests ??Confirmatory tests ?? To R/O dengue like illnessTo R/O dengue like illness Primary /Secondary infectionPrimary /Secondary infection

Lab investigations for confirmation of diagnosis Isolation of virus RT-PCRRT-PCR - considered as "gold-standard“ Serological tests -1)NS1 2)IgM/IgGSerological tests -1)NS1 2)IgM/IgG NS1 AntigenNS1 Antigen

1)Microwell ELISA TEST

2)Lateral Flow Rapid Test (LFRT) IgM/IgG

1) E/M-specific capture IgM and IgG ELISA

2) Rapid chromatographic test -IgM & IgG

NS1 antigen –Non structural proteinNS1 antigen –Non structural protein High concentration in the acute-phase of primary and

secondary dengue virus infections Up to 9 days after the onset of illness

NS1 testNS1 test ELISAELISA RAPID TESTRAPID TEST

Time requiredTime required 2 hr2 hr 15-30 min15-30 min

Cost/availabilityCost/availability Costly / +Costly / + Cheap / -Cheap / -

SpecificitySpecificity 100%100% 100%100%

SensitivitySensitivity 82%82% 72%72%

Primary/secondaryPrimary/secondary -- --

Basic immunologyBasic immunology

InterpretationInterpretation

Case - 2Case - 2

4hr old term male baby with maternal H/O 4hr old term male baby with maternal H/O PROMPROM

O/E O/E

C/C/A –Good C/C/A –Good

Clinical exam - NormalClinical exam - Normal Neonatal septic screeningNeonatal septic screening

2.Neonatal septic screening2.Neonatal septic screening

IndicationsIndications

1. Suspected sepsis

2. Preterm <34wks / wt <1800gm

3. PROM/Maternal fever/UTI

4. Respiratory difficulties

5. Temperature instability/fever

Neonatal septic screeningNeonatal septic screeningComponents Components 1.1. Total leukocyte count (<5000)Total leukocyte count (<5000)2.2. Absolute Neutrophil countAbsolute Neutrophil count3.3. Immature to mature ratio (I:T Ratio) >0.2Immature to mature ratio (I:T Ratio) >0.24.4. CRPCRP5.5. Micro ESRMicro ESR Septic screen is considered positive if Septic screen is considered positive if 2 or more 2 or more

parametersparameters are abnormal ( are abnormal (SensitivitySensitivity 93% 93% Specificity Specificity 83%)83%)

Septic screen does not include blood c/s Septic screen does not include blood c/s ,CXRay,or lumbar puncture –These are definitive ,CXRay,or lumbar puncture –These are definitive teststests

Sepsis screening – limitationsSepsis screening – limitations Interpret sepsis screen taking into consideration Interpret sepsis screen taking into consideration

the risk factors/gestation of infant/clinical the risk factors/gestation of infant/clinical status/timing of screenstatus/timing of screen

Taken isolation none of the tests are definite Taken isolation none of the tests are definite indicators of sepsisindicators of sepsis

Do not use septic screen as a substitute for Do not use septic screen as a substitute for blood cultureblood culture

Do not use SC to decide duration of antibiotic.Do not use SC to decide duration of antibiotic.

C Reactive ProteinC Reactive Protein

Produced by hepatocytesProduced by hepatocytes Healthy individuals <1mg dLHealthy individuals <1mg dL CRP starts to rise within 12 to 24 hours of onset

of sepsis (Earlier than the other acute phase reactants.)

Can rise 1000 fold within 24 hrsCan rise 1000 fold within 24 hrs Half life<24hrHalf life<24hr

CRP Practical points…CRP Practical points… Best studied in neonatal sepsisBest studied in neonatal sepsis Serial CRP levels are useful to exclude diagnosis of

neonatal sepsis. If two CRP measurements 24 h apart are <10 mg/L.--Negative predictive value >98%Negative predictive value >98%

Positive response needs correlation with other Positive response needs correlation with other parameters parameters -Positive predictive value <50%-Positive predictive value <50%

It is not justified to start the antibiotics only on the ground It is not justified to start the antibiotics only on the ground of positive CRP.of positive CRP.

Serial measurement provide additional information on Serial measurement provide additional information on the adequacy of treatmentthe adequacy of treatment

Trend of CRP is more important than a single CRP value in monitoring the activity of inflammation.

Micro-ESR

Simple marker for neonatal infection. Not a very reliable marker. Its normal value is 6 mm -first 3 days of life. End of first month, upto 11 mm. >15 mm is suggestive of infection.

Procalcitonin (PCT)

Procalcitonin is a reliable marker of lateonset sepsis in newborns with a sensitivityspecificity of almost 100%.

Case 3Case 3 7yr old boy – fever 6days7yr old boy – fever 6days Continuous - high grade - rising trend Continuous - high grade - rising trend D1-3 : No other localizing symptoms/signsD1-3 : No other localizing symptoms/signs D 4-5 –Vague abd pain ,loose stools D 4-5 –Vague abd pain ,loose stools D6 -Toxic ,104 F, Tongue - coated D6 -Toxic ,104 F, Tongue - coated P/A-Liver 2cms /span 8cms,Spleen 1cm / P/A-Liver 2cms /span 8cms,Spleen 1cm /

soft soft Provisional diagnosisProvisional diagnosis Enteric feverEnteric fever

CBC,Urine R/E, Blood c/sCBC,Urine R/E, Blood c/s Urine R/E –NormalUrine R/E –Normal CBC-TC-3400/cmm.CBC-TC-3400/cmm. DC-P 60 L 38 E 0 M 2DC-P 60 L 38 E 0 M 2 Hb 11.6 HCT 33.8Hb 11.6 HCT 33.8 Platelets 142000Platelets 142000

Clinical +Clinical +low leukocyte count with eosinopenia points to possible

Enteric fever

Blood culture -gold standard Sensitivity –highest in the first week of the

illness Reduces with advancing illness. Overall sensitivity - 50 % Drops with prior antibiotic therapy Bone marrow culture is a highly sensitive

even in late stages of the illness and with prior antibiotic therapy.

Blood c/s in practiceBlood c/s in practice Gold standard for bacteremias Gold standard for bacteremias IndicationsIndications Age <3mon with suspected infection Suspected Enteric fever FUO Febrile nutropenia/ Immuno compromised child Hospital acquired infectionPractical points Cleaning -70% Alcohol / 2% chlorhexidine Universal precautions Amount of blood – 1-5ml Blood : Media – 1:5 Preferably before antibiotics

Blood culture typesBlood culture types ConventionalConventional

LimitationsLimitations

1. Labor-intensive

2. Time consuming

3. Sub culture - each sample manually after 12,24,48 hr

Automated blood culture systemsAutomated blood culture systems1.1. Bacterial growth is detected by microbial production Bacterial growth is detected by microbial production

of COof CO22

2. BACTEC / BacT-alert / MGIT / BACTEC +

Automated culture

AdvantagesAdvantages Improved recovery of organismImproved recovery of organism Rapid isolationRapid isolation Better identificationBetter identification Accurate susceptibilityAccurate susceptibility

LimitationLimitation Non availability Cost

BETTER MANAGEMENT

Widal testWidal test

Widal test

Most widely used serological test Order this test only after 5-7 days of fever Tube method is better than slide method Both H and O antibodies of 1 in 160 dilution

should be taken as cut off value of diagnosis. H antibodies once positive can remain positive

for long time.

Serum Widal –Limitations…. Timeline - Send only after first week of fever Poor sensitivity- false negative 30% Previous antibiotic treatmentPrevious antibiotic treatment Poor specificity - endemicity & anamnestic reasons Method : slide test – Not reliable VaccinationVaccination whole cell vaccine-High baseline anti-o &anti-H whole cell vaccine-High baseline anti-o &anti-H (Vi-polysaccharide vaccine does not interfere ) Inter laboratory variations

Typhidot test

A dot enzyme immunoassay that detects specific IgM and IgG antibodies - 50KD outer membrane protein antigen of S. typhi.

Limitation: False positive results in endemic areas.

-Persistence of high IgG levels Typhidot-M® - only IgM is detected. IgG is

inactivated Typhidot-M® -sensitivity (>93%)

Molecular methods

PCR for Typhoid Superior sensitivity than culture The turnaround time < 24 hours

Limitations Clinical utility - inadequately evaluated False positive –Vaccination , Old infections Costly

Tubex test: Tubex test is simple one-step test taking 2minutes It detects only IgM and not IgG Positive result -Suggests recent Salmonella

infection

IgM dipstick test Based on the binding of S. typhi-specific IgM

antibodies to S. typhi lipopolysaccharide (LPS) antigen.

It can be done in serum or whole blood and requires incubation for 3 hours

Case-4Case-4

4yr old girl -H/O High grade fever 3 days4yr old girl -H/O High grade fever 3 days Sick looking. No focus of infectionSick looking. No focus of infection Poor response to symptomatic treatmentPoor response to symptomatic treatment Admitted for evaluation Admitted for evaluation Supportive treatmentSupportive treatment Investigations ? Investigations ? CBC, Urine R/E CBC, Urine R/E

Case 4– Lab reportCase 4– Lab report

CBCCBC TC-23,000/cmm DC :P 82 L 19 E 0 M 1TC-23,000/cmm DC :P 82 L 19 E 0 M 1 Hb 11 gm % HCT 33.7 Hb 11 gm % HCT 33.7 Platelets 3,31,000Platelets 3,31,000

Urine analysisUrine analysis Protein +Protein + Leucocyte esterase + Nitrite +Leucocyte esterase + Nitrite + Microscpy 40-50 / hpfMicroscpy 40-50 / hpf

Diagnosis: ?UTIDiagnosis: ?UTI

Mild proteinuria Significant pyuria >10 leukocytes per mm in a

fresh uncentrifuged sample, or >5 leukocytes per high power field in a centrifuged sample.

Bacteria on Gram staining

Dipstick

Leucocyte esterase test –Pyuria

Nitrite test - significant Bacteriuria

Leucocyte esterase + Nitrite - more sensitive

Clinical application…Clinical application… Useful in screening for UTI.

Combination of these tests has moderate sensitivity and specificity for detecting UTI

Positive nitrite + leucocyte esterase ( sensitivity 72% and specificity 96%)

Positive Gram stain (sensitivity 93% and specificity 95%)

Urine c/sUrine c/s The diagnosis of UTI is based on positive

culture of a properly collected urine. Collection & storage of sampleCollection & storage of sample Sample must be obtained prior to therapy

with antibiotics Midstream specimen Urinary bags Suprapubic aspiration Urethral catheterization is reserved for

unconscious patients already catheterized. Ensure prompt plating or storing at 4c upto 24 hours after urine collection

Urine culture Interpretation

Radio imaging studies• Recommended for all children with UTI.

• Aim - to identify patients at-risk of renal damage, mainly those below 5 years of age, with VUR or urinary tract obstruction.

USG in UTIUSG in UTI

USG to be done in all cases of UTIUSG to be done in all cases of UTI

To L/FTo L/F

1.1. Posterior urethral valvePosterior urethral valve

2.2. Post void residual urinePost void residual urine

3.3. Bladder hypertrophyBladder hypertrophy

4.4. HydronephrosisHydronephrosis

5.5. Pelvicalyceal anomaliesPelvicalyceal anomalies

MCU ScanMCU Scan

It will show anatomy of the bladder and the It will show anatomy of the bladder and the ureters.ureters.

It is basically for bladder and VUR issuesIt is basically for bladder and VUR issues

A. The MCU showing adilated posterior urethra, mildly irregular appearance of theedge of the bladder and bilateral vesicoureteric reflux into dilated tortuous ureters.

B. Hydronephrosis with 'clubbing' of the calyces.

DMSA ScanDMSA Scan Gold standard test for detecting renal cortical Gold standard test for detecting renal cortical

scarsscars Can be done during acute phase of UTICan be done during acute phase of UTI

Case 5: Fever with rashCase 5: Fever with rash 3 yr old girl with fever – 4 days , 3 yr old girl with fever – 4 days , Skin rash – 2 daysSkin rash – 2 days Fever-moderate-highFever-moderate-high Rash increased over 2 days and lead to black Rash increased over 2 days and lead to black

patches over one ear lobe and one toepatches over one ear lobe and one toe

O/EO/E Sick child with high feverSick child with high fever Maculonodular rashMaculonodular rash Gangrenous patches-Ear lobe, one toeGangrenous patches-Ear lobe, one toe

Clinical diagnosis…Clinical diagnosis… Rickettsial diseaseRickettsial disease Investigation Investigation No rapid laboratory tests are available to No rapid laboratory tests are available to

diagnose rickettsial diseases early in the diagnose rickettsial diseases early in the course of illness.course of illness.

Gold standard –(Gold standard –(IFA )Immuno fluorescence assay IFA )Immuno fluorescence assay Weile Felix - Useful and cheapest available tool for Weile Felix - Useful and cheapest available tool for

the laboratory diagnosis of Rickettsial diseases in the laboratory diagnosis of Rickettsial diseases in India India

ELISA / RT-PCR / ELISA / RT-PCR / dot blot immunoassay dot blot immunoassay

Weil-Felix Weil-Felix is a nonspecific agglutination test is a nonspecific agglutination test It detects anti-rickettsial antibodies in patient’s It detects anti-rickettsial antibodies in patient’s

serum. serum. If the  titer is greater than or equal to 1:320 or If the  titer is greater than or equal to 1:320 or

4-fold rise from baseline - 4-fold rise from baseline - POSITIVEPOSITIVE sensitivity(33%) and specificity(46%)sensitivity(33%) and specificity(46%)

IFA -Immunofluorescence assayIFA -Immunofluorescence assay

Gold standard for diagnosisGold standard for diagnosis IFA can detect IgG or IgM antibodies. Blood samples taken early (7-10days) and late

(14-21days) in the disease are the preferred specimens for evaluation.

Rising titres of IgM is indicative of a current infection

Case 6Case 6 5yr old boy – Frequent cough cold & fever – 1yr5yr old boy – Frequent cough cold & fever – 1yr 6 episodes /yr 6 episodes /yr Each episode -2-4days of fever Cough&cold-5-10 dEach episode -2-4days of fever Cough&cold-5-10 d No hospitalizations/No family h/o AtopyNo hospitalizations/No family h/o Atopy Clinical exam - NormalClinical exam - Normal Inv-Hb 11g% TC 14300 P35 L62 E3 ESR 42 mmInv-Hb 11g% TC 14300 P35 L62 E3 ESR 42 mm Mx test- 10 mm PositiveMx test- 10 mm Positive. . Rx –ATT – Referred for 2Rx –ATT – Referred for 2nd Opinion nd Opinion

No h/o contact , X – Ray chest –NormalNo h/o contact , X – Ray chest –NormalDiagnosisDiagnosis Recurrent viral respiratory infectionRecurrent viral respiratory infection

Tuberculosis diagnosisTuberculosis diagnosis

Golden triadGolden triad

(Clinical features +Abnormal chest X-ray +Mx)(Clinical features +Abnormal chest X-ray +Mx)

++ contact history/close exposure with an adult having contact history/close exposure with an adult having active TB in the last 2 yearsactive TB in the last 2 years

Demonstration of AFBDemonstration of AFB - Confirmatory diagnosis - Confirmatory diagnosis

AFB in smear/cultureAFB in smear/culture Isolation of MTBIsolation of MTB –Clinical specimen/histopathology –Clinical specimen/histopathology

Mantoux testMantoux test

ID - PPD-2TU- 2-4 inch below ID - PPD-2TU- 2-4 inch below elbowelbow

Transverse diameter of the Transverse diameter of the induration (not erythema) induration (not erythema) measured- 48 -72 hours in mmmeasured- 48 -72 hours in mm

Positive >10mm-InfectionPositive >10mm-Infection Reaction could be due to

1. Infection with tubercle bacilli

2. Cross sensitivity to environmental mycobacteria

3. BCG-induced sensitivity

Mantoux test interpretationMantoux test interpretation

TB diagnosis- Newer methods

1. Direct molecular methods

2. Automated liquid culture methods

Automated liquid culture

BACTEC TB 460 Sensitive, specific and rapid culture method Respiratory /non-respiratory specimens. 200 viable M. tuberculosis bacilli could be detected in

12-13days

Mycobacterial growth indicator tube(MGIT) 960 TB: Fluorescent technology - based on oxygen quenching

with a fluorescent dye. Result - 7-10 days.

Direct molecular methods

Do not require growth of the bacteria, Possible to detect M.tuberculosis complex within

3-5 hours. Nucleic Acid Amplification assay tests

1. The Gen-Probe AMPLIFIEDTM Mycobacterium tuberculosis Direct (MTD) Test.

2. The Roche AMPLICOR® Mycobacterium tuberculosis (MTB) Test.

Limitation Results of NAA tests are preliminary; the

mycobacterial culture is needed for species identification, confirmation and for drug-susceptibility testing.

A positive result may be misleading as the NAA test can amplify DNA from both viable and non-viable organisms

Case 7Case 7

5 yr old boy with fever 5days5 yr old boy with fever 5days High grade, intermittent, Chills & rigors +High grade, intermittent, Chills & rigors + Headache ,Vomiting +Headache ,Vomiting + O/E Temp 103 FO/E Temp 103 F P/A Spleen measuring about 3 cm below costal margin P/A Spleen measuring about 3 cm below costal margin

– firm , non-tender– firm , non-tender No hepatomegalyNo hepatomegalyDiagnosisDiagnosisMalariaMalaria

Lab investigationsLab investigations

Lab: Hb 11.2 TC 13500 P72 L26 M2Lab: Hb 11.2 TC 13500 P72 L26 M2 MP smear – Plasmodiam vivax ring ,gametocytesMP smear – Plasmodiam vivax ring ,gametocytes

MalariaMalaria

Diagnostic techniques

1. Blood smear examination- gold standard

2. Quantitative buffy coat technique (QBC)

3. RDTs

4. Fluorescent microscopy

Blood smear for malarial parasiteBlood smear for malarial parasite

Thick blood smears are most useful for detecting the presence of parasites, because they examine a larger sample of blood.

Thin blood smears helps doctors discover what species of malaria is causing the infection.

Disadvantages of MicroscoprDisadvantages of Microscopr

Time consuming >60minTime consuming >60min Labour intensiveLabour intensive Skilled lab technicianSkilled lab technician It cannot detect parasites sequestered deep in It cannot detect parasites sequestered deep in

the vascular compartmentthe vascular compartment

QBC for MPQBC for MP The blood is taken in a QBC The blood is taken in a QBC

capillary tube which is coated with capillary tube which is coated with acridineacridine orange orange (a fluorescent dye)  (a fluorescent dye) and centrifugedand centrifuged

The The fluorescingfluorescing parasites can then  parasites can then be observed under be observed under ultravioletultraviolet light  light at the interface between red blood at the interface between red blood cells and buffy coat. cells and buffy coat.

This test is more sensitive than the This test is more sensitive than the conventional conventional thick smearthick smear and  and

> 90% of cases the species of > 90% of cases the species of parasite can also be identified. parasite can also be identified.

Rapid tests for malaria Immunochromatographic tests based on the

‘Dipstick’ format Detects plasmodium specific antigens in blood

sample. Common RDTs available Parasight ,OptiMAL.

Targeted antigens Histidine rich protein II of P. Falciparum (pfHRP-II) Plasmodium aldolase- produced by all plasmodium

species. Plasmodium lactate dehydrogenase(pLDH), is

produced by all four species of plasmodia. Antibodies produced against pLDH either specific for

P.falciparum or P.vivax alone or a pan specific antibody which reacts with all the four species of plasmodium. Commercially available kit can detect falciparum and vivax but cannot differentiate ovale and malariae.

Advantages Rapid tests are easy to use with minimal training Results are available within minutes. They can diagnose falciparum infection even

when the parasite is deeply sequestered.

Disadvantages More expensive than microscopy Limitations in species identification. Persistent positivity even after effective

treatment. HRPII remains positive for 4 weeks and pLDH remains positive for1 week.

No prognostic value- they can not quantify parasites.