Jpn. J. Infect. Dis., 54, 237‑239, 2001

Short Communication

Utility of PCR in Diagnosis of Problematic Cases of Typhoid
Abdul Haque*, Naeem Ahmedl , Anwar Peerzadal , Abida Raza,Samina Bashir and Ghulam AbbasI
National Institutefor Biotechnology and Genetic Engineering (NIBGE), Jhang Road andJMedical Wwd, Allied Hospital, Punjab Medical College, Jail Road, Faisalabad, Pakistan(Received October 9, 2001. Accepted December 25, 2001)

SUMMARY: Typhoid is a global problem. Conventional diagnostic methods have limitations. The Widal test
gives a high proportion of false positive results, and indiscriminate use of antibiotics has reduced the utilityof
blood culture･ Consequently, these procedures are inadequate fわr diagnoslng Suspected cases or typhoid that do

not present clear‑cut symptoms･ We previously showed that PCR‑based diagnosis of typhoid targetlng theflagellin
gene has unparalleled specificity･ We assessed the utility of this method for diagnosis of problematic cases oftyphoid･ A comparative study of PCR, blood culture, and Widal test was ca汀ied out on 55 cases of suspectedtyphoid with fever for 3‑30 days and possessing an ambiguous clinical picture. A control group comprised of 20
healthy persons was also included. The respective positive results by PCR, blood culture, and Widal test f♭rthese groups were 58･2 and 0%, 14･5 and 0%, and 52･7 and 45%･ Sensitivity ofPCR as coヮpared with that ofblood culture was slgnificantly better･ We concluded that PCR is much superior to conventional methods and,due to its high sensitivityand specificity, can be of greatuse for rapid and definitive diagnosis of problematiccases of typhoid.

There are more than 2 1 million c?ses of typhoid each year

In this study, suspected cases of typhoid with fever for 3‑

throughout the world (1 ). h developlng countries, due to poor

30 days but an ambiguous clinical picture (only one or twohygienic conditions and inadequate medical facilities,classical symptoms such as enlarged spleen, headache, rose

subclinical infection is very common. Many microbiologlCal

spots, malaise, abdominal discomfort, lethargy, constipationand serological techniques have been used for diagnosis offollowed by diarrhea, fatigue, delirium, and agitation) werethese cases. These techniques include blood culture, bone

included･ Patients represented both sexes and a wide range

marrow culture, rectal swab culture, urine culture, rose spotor ages. The control group consisted of 20 healthy individuals

culture (2‑3); duodenal string culture (4); Widal test (5‑6);

with no history of fTever for the previous year. PCR (targetingand ELISA (5).
Widal test and blood culture remain the most common

theflagellingene), blood culture, and Widal test were performed

diagnostic procedures because other methods are either

specificity, and sensitivlty Of these methods.

on samples from these cases to evaluate the relative utility,invasive or they have failed to prove routinely utile. Widal

Two well‑characterized strainsof Salmonellaりphi (STK‑ 1

test is sensitive but highly nonspecific (6,7). Therefore, its

and STK‑2) Were used f♭r standardization of PCR conditions,effectiveness in diagnosis of varied cases of typhoid is notand as reference strains. Extraction of DNA from bacterial

slgnificant. Blood culture, though highly specific, is helpful

cultures and blood samples was perfわrmed by the phenol/

only in diagnosis ofearlytyphoid. In such cases, it can only

chloroform method as previously reported (1 2)･ For blood

detect 40‑45% of positive subjects (8).

culture (perf♭med on two successIVe days), 2 ml orblood

Hybridization uslng DNA probes, the first molecular biology

was inoculated into a culture bottle containlng 16 ml oftechnique used, has 99% specificity but cannot detect lesstrypticase soy broth (Difco, Detroit, Mich., USA) with 0.02%

than 500 bacteria/ml (9). However, the advent of PCRSPS (sodium polyanethanol sulfonate) and incubated at 37oC
for 72 h. Using the broth culture, subcultunng and biochemical
identification was performed by conventional methods (1 3).

technology, already in use fわr various other diseases, hasprovided a powerful diagnostic tool. Even I‑5 bacteria/ml
are detectable, and results can be available in 1‑2 days (10‑Confirmation of isolates was carried out by PCR. Widal test12).

was performed by tube method according to manufacturer's

In developlng countries, usually 3‑4 days after infection

instructions, using reagents provided by Biosystems (Barcelona,elapse before the patient grows concemed about his or herSpain).

protracted fever. Such cases can present a challenge to theFor PCR, primers reported by Song et al. (1 1) and modified

clinician･ The clinical symptoms are usually vague, andby Frankel (14), which target theflagellin gene ofS.りphi,routine diagnostic methods have limited utility. This study

were synthesized on Phamacia LKB Biotechnology (Uppsala,was designed to evaluate the value ofPCR f♭r diagnosis inSweden), Gene Assembler Special. 01igonucleotides STl (5′‑such cases.

TATGCCGCTACATATGATGAG‑3 ') and ST2 (5 '‑TTAACGCAGTAAAGAGAG‑3 ') were used for regularPCR to amplify*Corresponding author: Mail address: Health Biotechnology

a 495 bp fragment corresponding to nucleotides 103611056

Division, National Institute fわr Biotechnology and Genetic

and 15 13‑1530, respectively, in the nagellin gene ofS.りphi.

Engineering (NIBGE), P.0. Box 577, Jhang Road, Faisalabad,

For nested PCR, oligonucleotides ST3 (5′‑ACTGCTAAAA

Pakistan. Tel: +92‑41‑651475‑79 Ext. 225/242, Fax: +92‑41‑

CCACTACT‑3 ') and ST4 (5 '‑TGGAGACTTCGGTCGCGT

65 1 472, E‑mail: abdulhaq@fsd.comsats.net.pk

AG‑3 ') were used to amplifya 363 bpfragment corresponding237

to nucleotides 1072‑1089 and 141611435, respectively, of thePCR and blood culture were negative in all cases. However,same gene. For regular PCR, loo LEI DNA amplification

Widal test was positive 45% of cases.

mixture contained 10 mM Tris HCl (pH 8.3), 50 mM KCl,
1.5 mM MgC12, 0.01% gelatin, 100 pmol of each prlmer, 70

Typhoid is a major disease in developlng countries. Routine
efforts are made to treat patients by self‑medication facilitatednmol of each dNTP, 1 LEI ofAmpli Taq, 20 LEI oftemplate,and distilled water to make up the volume. Using a thermalcycler (Perkin‑Elmer Gene Amp PCR system 2400: Perkin‑
Elmer Corp., Norwalk, Conn., USA), the reaction mixture
was subjected to 25 cycles of I min each at 94oC, 55oC, and72oC, followed by heating at 72oC for 7 min. For nested PCR,

by the free availability of drugs without medical prescrlP‑

conditionswere the same except that a I :5 dilution of amplified

for typhoid diagnosis because they can be easily carried out

product was used as the template. PCR was performed inand patient's comfわrt is not compromised. Widal test is non‑tion. By the time the clinician is consulted, usually at least 3‑4days have passed. This delay not only makes clinical diagnosis
di爪cult in many cases, but renders conventional diagnostic

methods inadequate or useless.

Widal testand blood culture are the most favored teclmiques

duplicate fわr each sample. A positive control representlng 5

specific, and false positive. results are very common (15)･

bacteria/ml of S.りphi and a negative control without any

Blood culture has the promlSe OfdiagnosIS in the丘rst week

DNA werealso included in each lot. Ten microliters of reaction
mixture was fractionated electrophoretically in 2.5% agaroseof infection, but sensitivlty lS poor. Less than 1 0 bacteria/ml
can cause severe infection, and are usually undetectable byblood culture (16). At later stages, the sensitivity ofblood

gel containlng 0.5 〝g of ethidium bromide per ml and wasculture declines even further as a result of some limltlng

photographed using Eagle Eye (Stratagene, La Jolla, Calif･,USA).

factors such as the host's immune response system and theintracellular characteristics of S.りphi (1 7).

It has been reported that a single bacteriuln Of S.りphicontains 4 fg ofDNA (1 1). Using this reference, dilutions
were made in distilled water from DNA extracted &om cultureof standard strains STK‑ 1 and STK‑2, then used as the templatefor PCR. The results showed that the regular PCR had asensitivity of 105/ml, whereas nested PCR could detect even

PCR has been reported to detect even I‑5 bacteria/ml withhigh specificity(10, 12). We thought that it would be especially
useful for diagnosis of problematic cases of typhoid in which
the patient comes late to the clinician and does not havespecific clinical symptoms. We selected 55 such cases inwhich patients had experienced fever for 3‑30 days. Widal

5 bacteria/ml (data not shown).

test, blood culture, and PCR were performed on blood samples

The patients suspected of typhoid included in this study
had experienced fever for 3‑30 days, With suggestion of斤om these patients. A control group of 20 healthy individuals

typhoid fever but no clear‑cut diagnosis.Asshown in Table I,
Widal test wTs positive in 52･7% (29/55), and blood culture

having no recent history of fever was also included.

was positive ln Only 14.5% (8/55) of cases. PCR was positivein 58.2% of cases (32/55), and among these, 14.5% (8/55)

its hypervariable region Vl isunique for S.りphi (1 1 , 14). There

For PCR, we preferred to target theflagellin gene because

is some controversy over targeting this gene, and the specificityof this method has been discussedinvanousforums. However,were positive for PCR only. Therefore, had the PCR not been

performed, these eight cases would not have been diagnosed･

The results ofPCR, blood culture, and Widal test performed

Fs pointed out by Frankel (14), if a different forward primer
lSused for first PCR which is derived from the DNA sequenceon healthy persons reportlng nO recent history of fever areJust upstream Ofprimer STl , together with ST2 as describedshown in Table 2. This group wasused as a negative control.

by Song et al. (1 1), only S.妙hi and S. muenchen sequences
are amplified. The use of STl, which is S.りphi speciflC,together with ST4 for nested reaction gives I 00% speciflCity･
The altemative method, in which the n gene is targeted, canTable 1. Comparative study of PCR, blood culture, and Widal test in

suspectcd cases oftypohoid

give false positive results because of the presence of S.

paraophi C (10). We were able to improve the sensitivity ofNo. of cases PCR Blood culture Widal tcstl′0 2 ′hV O 7 ∠U

our nested PCR to a detection level of less than 5 bacteria/ml+++++(data not shown). Our previous experience with this technique(12) and our experience in this study suggest that it is a verysatisfactory method for diagnosis of typhoid.

+l

Our results show that Widal test had no discriminatlng+power, glVen血at 45 and 52.7% of cases ofhealthy individuals

and febrile patients, respectively, showed positive. Respective
55

32(58.2)28(14.5)

29(52.7)

values for blood culture were 0 and 14.5%, indicating a very
high level of specificitybut poor sensitivity. PCR values forthese groups were 0 and 58.2%. Comparatively, lt is qulte

IWidal test was considered positive if titer of antibodies against
Salmonella坤hi 0 antlgen Was more than I : 1 60.

2Figurcs in parenthcscs indicatepercentagc.

clear that PCR is much superior to these tests in sensitivity

and specificity. The importance of PCR in diagnosis of prob‑
lematic cases of typhoid is further highlighted by the observa‑

Table 2. Comparative study ofPCR, blood culture, and Widal test intion that eight (14･5%) case! were positive only for PCR･ andhcalthy individuals

these cases would been mlSSed by either of the other twoNo. of cases PCR Blood culture Widaltcstl

methods.
Generally, there have been some reseⅣations about the useof PCR for diagnosis of typhoid. However, our present study
2000

clearly shows that targetlng the nagellin gene by careful

9(45)2

I Widal test was considered positive if titcr of antibodies against

selection of primers makes it an excellent tool for diagnosis ofSalmonella Ophi 0 antigen was morethan 1 : 160.

problematic cases of typhoid. Given that PCR‑based diagnosis

2 Figures in parentheses indicate percentage.

of infectious diseases like tuberculosis and hepatitis B and C

238

diagnosis of typhoid. Infect. Dis. J., 3, 19･

has proven its value in recent years, the diagnostic power ofPCRfor difRcult cases of typhoid, a very serioushealth problem

8. Guerra‑Caceras, ∫. G., Gotuzzo‑Herenicia, E., Crosby‑Dagnini, E., MiroIQuesada, J. and Cari110‑Parodi, C. ( 1 979):

in developlng COuntries, holds great potential fわr use･

Diagnostic value of bone marrow culture in typhoid

fever. Trans. R. Soc. Trop. Med. Hyg., 73, 6801683.
9. Rubin, F. A., McWhirter, P. D., Punjabi, N. H., Laane, E.,
Sudamono, P., Pulungsih, S. P., Lesmana, M., Kumala,S., Kopecko, D. J. and Hoffman, S. L. (1989): Use ofa

ACKNOWLEDGMENTS
We are grateful to Dr. Ahmed Mukhtar Khalid, Director,
National Institute for Bioteclmology and Genetic Engineerlng(NIBGE), Faisalabad, and Dr. Javed Anver (〕ureshi, Head,DNA probe to detect Salmonellaりphi in the blood ofHealth Biotechnology Division of the same institute, for theirpatients with typhoid fever. J. Clin. Microbio1., 27, 1 I 12‑1114.

guidance and advice.

10. Hashimoto, Y., ltho, Y., Fujinaga, Y., Khan, A. Q., Sultana,F., Miyake, M., Hirose, K., Yamamoto, H. and Ezaki, T.

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