Diagnostic Usefulness of Loop - mediated Isothermal Amplification for Intrafamilial Pertussis Infection

As a Bordetella pertussis gene test, the loop-mediated isothermal amplification (LAMP) method has become widely used for the diagnosis in clinical practice. The diagnostic usefulness of the LAMP method was investigated in 11 infant patients and their 12 families retrospectively, who were judged as having intrafamilial pertussis infection. Eleven pediatric patients (Index cases) and their 26 family members (10 families) who developed cough and characteristic symptoms of pertussis, that persisted for 2 weeks or longer and were clinically diagnosed as having pertussis between May 2010 and February 2013, 37 patients in total, were investigated. Thirty-three of 37 patients were definitively diagnosed with pertussis and 31 of them (83.4%) were LAMP-positive. Culture and LAMP tests were simultaneously performed in 10 families (26 patients). Among these families, all infants (11 patients) were LAMP-positive, 8 of their siblings, and 10 parents were LAMP-positive, and the positive rate was higher than the culture positive rate. Since LAMP positivity persists for a relatively prolonged period after disease onset, adult patients with atypical symptoms can be diagnosed and the source of pertussis infection in the family can be clarified. The disease through silent transmission may become aggravated when infants have received DTP vaccine. The positive rate on the LAMP test for Bordetella pertussis was higher than that on the culture test, after 4 weeks or more after the onset. It should be examined for the asymptomatic family members in the case of the pertussis onset. Because pertussis infection cannot become clear only for a symptom. Additional vaccination of pertussis for adult is necessary even though vaccination has been completed in infancy.


Introduction
Large-scale pertussis outbreaks occurred in Japan in 2007. Thereafter, the proportion of infected adults increased, and adults aged 20 years or older account for half of pertussis patients 1) . Intrafamilial infection has been problematic as a source of infection of infantile pertussis. As a Bordetella pertussis gene test, the loop-mediated isothermal amplification (LAMP) method became covered by national health insurance in October 2016 in Japan and has become widely used for the diagnosis in clinical practice. In this study, the diagnostic usefulness of the LAMP method was investigated in 11 infant patients and 10 families, who had been determined as having intrafamilial pertussis infection.

Patients
Eleven pediatric patients (Index cases) and their 26 family members (10 families) who developed cough and characteristic symptoms of pertussis, that persisted for 2 weeks or longer and were clinically diagnosed as having pertussis between This study protocol was reviewed and approved by the Ethics Committee of Koshigaya Municipal Hospital (Approved No. 30-4). Oral informed consent was obtained.

Methods
Pertussis was definitively diagnosed when one of the following conditions was met: 1) Bordetella pertussis was isolated on culture of the nasal posterior nares. Bordetella CFDN agar medium was used for culture of the Bordetella pertussis. The medium plates were incubated at 35℃ in a humectation condition. 2) B. pertussis was detected in nasal discharge, posterior nares, or sputum using the LAMP method (Loopamp Bordetella pertussis detection kit, Eiken Chemical Co., Ltd., Tokyo), and 3) serologically, the PT-IgG level was 94 EU/ml or higher in single serum or 2 times or more increase was detected in paired sera 2) . Bordetella antibody EIA (DENKA SEIKEN Co., Ltd) for the test of PT-IgG was used. Among the subjects, the culture, LAMP, and PT-IgG tests were performed in 30, all 37, and 17 patients, respectively.

Results
Nine, 29, and 5 of the 37 patients were positive for the pertussis tests by culture, LAMP, and PT-IgG, respectively. Overall, thirty-three patients were positive, with a positive rate of 89.2%. Twenty-nine (93.5%) of the 31 patients were LAMP-positive. Of the Infants, 6, 11 (100%), and 2 patients were positive for the pertussis tests by culture, LAMP, and PT-IgG, respectively. Of the 11 index cases, one patient had received step I of the DPT vaccine once, 2 patients had completed additional step I vaccinations, 5 patients were not vaccinated, and the history of vaccination was unclear in 3 patients. Excluding one 9-month-old patient from among the nonvaccinated patients, 4 patients were younger than 2 months old, which were not included in the indication for DTP vaccine. Of the siblings definitively diagnosed with pertussis, 1, 8, and 2 were culture-, LAMP-, and PT-IgG-positive, respectively. Of the parents definitively diagnosed with pertussis, 2, 10 (71.4%), and 1 were culture-, LAMP-, and PT-IgG-positive, respectively.
Ten families (26 patients) who were simultaneously examined by culture and LAMP and definitively diagnosed were investigated (Figure-1). Of the 11 index cases, 6 (54.5%) were positive on the culture test, whereas 11 (100%) were positive on the LAMP test. Of the 12 siblings, 1 (11.1%) and 8 (66.7%) were positive on the culture and LAMP tests, respectively. Of the 14 parents, only 2 (14.3%) were positive on the culture test, but 10 (71.4%) were positive on the LAMP test.
The clinical symptoms were investigated in 7 families (16 patients) who were negative on culture and diagnosed by LAMP. Cough was noted in 13 (81.3%) of the 16 patients. Remaining three people did not have a symptom associated with pertussis including rhinorrhea. Repetitive cough was noted in 6 (37.5%) of the 16 patients. Cyanosis was noted in 2 index cases and apnea was noted in one of them. Cough was noted before the index case developed pertussis in 9 of the 10 families. Cough was noted in all 11 Index cases, but it was noted in 11 of the 12 siblings and 9 of the 14 parents. The time to the test from the onset of cough was 1-4 weeks (mean: 1.5 weeks) in the index cases, 1-8 weeks (mean: 2.2 weeks) in the siblings, and 0-6 weeks (mean: 2.0 weeks) in the parents.
In the 37 patients examined by the LAMP test, the positive rate was compared between those within 3 weeks after onset (23 patients) and at 4 weeks or later after onset (6 patients) (Figure-2). In the 29 patients examined by the culture test, the positive rate was 38.4% (8/23) within 3 weeks and 16.7% (1/6) after 4 weeks. The LAMP test positive rate was 80.6% (25/31) within 3 weeks and 66.7% (4/6) after 4 weeks. The PT antibody titer was measured in 17 patients and the positive rate was 15.4% (2/13) within 3 weeks and 75% (3/4) after 4 weeks.

Discussion
The highest positive rate was achieved by the LAMP method, with 29 (78.4%) of the 37 patients, showing a favorable sensitivity. Since the PT-IgG antibody titer is influenced by vaccination, paired sera samples are necessary to make a definitive diagnosis. The gold standard method for pertussis diagnosis is the culture test; however, there are problems with this test, as the positive rate is low, special media is necessary, and it takes 7 days to detect colonies, which is longer than the time required for a normal culture test. The Nucleic acid Amplification Test (NAT) has been available as a highly-sensitive test method and the polymerase chain reaction (PCR) method has been employed; however, its procedure is complex, takes time and can be expensive. Although the LAMP method is also a nucleic acid amplification test, the procedure is simpler than that of PCR and it is quicker. The LAMP method targets the pertussis toxin promoter region (ptxP) 3) . Since the ptxP of Bordetella pertussis is different from that of the other Bordetella species, it can be detected with high sensitivity; the sensitivity and specificity of LAMP for B. pertussis are 83 and 95%, respectively 3) .
Of the 26 patients diagnosed with pertussis based on the culture and serum tests, 19 (73%) were LAMP-positive in another report 4) , which is consistent with the high LAMP positive rate in our study. While all Index cases were LAMP-positive, the positive rate in the siblings and parents was about 70%. It was considered that the positive rate was related to the amount and timing of sampling Bordetella pertussis. In the present study, a prolonged duration had passed after the onset of the source of infection in the family by the time the index case developed the disease and was subjected to the tests. The tests were performed 1.5 weeks after onset in the index cases, whereas the tests were performed 2.0-2.2 weeks after onset in the siblings and parents; a little later. For the pertussis test, CDC recommends serological diagnosis when more than 4 weeks have passed after disease onset 5) . When differences in the positive rate due to timing were investigated, the culture test positive rate was low within 3 weeks after the onset. In contrast, more than 90% were positive within 3 weeks on the LAMP test and the rate was 60% even after 4 weeks. The PT-IgG antibody titer positive rate was high after 4 weeks.
The LAMP test, PT-IgG and culture test were investigated in pediatric cases by Sakamoto et al. 4) . This study was examined in familial cases including the difference of the pertussis positive rate of each group.
On the PCR test, pertussis positivity persists for a long time, even after treatment in some cases 6) , suggesting that the sensitivity of the LAMP test as a NAT test is also high. Since the LAMP test is more sensitive than conventional PCR 7) , it may be useful in the diagnosis of pertussis infection.
Intrafamilial pertussis infection is problematic for neonates and early infants. Typical symptoms do not develop and only protracted cough occurs in children who have received the pertussis vaccine and adults. Pertussis infection is not discovered until typical symptoms develop in some infants and young mothers with close contact with children are at high-risk 8) . In addition, it has been reported that the source of infection of infants admitted for pertussis was vaccinated elder siblings and grandparents. Since all members of the family may serve as a source of infection, prevention of horizontal transmission of pertussis by early diagnosis is necessary. In the present study, 4 parents of intrafamilial cases did not develop cough despite being LAMP-positive, and one of them was a mother who was positive on both LAMP and culture. It has been reported that the bacteria are excreted in the absence of any typical airway symptoms of pertussis in 14-20% of families 9) 10) . Since children vaccinated in infancy and adults can serve as an asymptomatic source of infection 11) , additional administration of pertussis vaccine is necessary even though vaccination has been completed in infancy. All 9 parents were LAMPpositive but 3 of them did not develop cough. It should be examined for the asymptomatic family members in the case of the pertussis onset. Because pertussis infection cannot become clear only for a symptom. The LAMP examination for pertussis might be more useful to search the onset in the family.

Conclusion
The positive rate on the LAMP test for Bordetella pertussis was high compared with that on the culture test even, after 4 weeks or more after the onset. Additional vaccination of pertussis for adult is necessary even though vaccination has been completed in infancy.